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beta-Amyloid-induced toxicity in rat hippocampal cells: in vitro evidence for the involvement of free radicals.

The conditions under which amyloid is toxic to primary rat hippocampal neurons were investigated. Synthetic A beta (1-42) peptide elicited neurotoxic activity following "aging" for 7 to 14 days at 37 degrees C in Modified Eagles Media. Neurotoxicity included decreases in neurite length, cell number, and a loss in 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide reduction. In contrast, the addition of the media supplement B27, during the aging process, promoted the neurotrophic actions of aged A beta (1-42), as indicated by an increase in neurite length and the number of cells possessing neurites, and attenuated toxicity. The differences in the biological actions elicited by these two preparations of aged peptide were attributed to the presence of the B27 components. B27 consists of a mixture of agents that provide protection against oxidative damage. In support, aging A beta (1-42) in the presence of superoxide dismutase and catalase, two components of B27, significantly reduced the toxic actions of peptide; hence, suggesting that free radicals may be required for the toxicity that accumulates during the aging of the peptide. To determine the contribution of particular amino acid residues in amyloid toxicity, studies were carried out with an aged preparation of the A beta (1-42) analog, A beta (1-42)Nle35. Findings from these studies suggest that the methionine residue may play a part, but is not required, for amyloid toxicity to occur.

Alzheimer Disease↗

Can 29kDa rhGM-CSF expressed by silkworm pupae bioreactor bring into effect as active cytokine through orally administration?

In order to study the effect of human granulocyte-macrophage colony-stimulating factor (hGM-CSF) as active cytokine through orally administration, we expressed hGM-CSF within silkworm pupae bioreactor. The purified rhGM-CSF named as BmrhGM-CSF is characterized as 29kDa glycoprotein, and its biological activity was measured both in vitro and in vivo. We found out BmrhGM-CSF could stimulate the colony formation of human bone marrow cells in a dose-dependent manner whether which were treated with or without gamma-ray 24h before. The ability of colony formation induced by BmrhGM-CSF is negatively correlated with gamma-ray intensity. As soon as 15min post oral administration with BmrhGM-CSF labeled with (125)I, an approximately 20kDa protein fragment was detected within mice blood by SDS-PAGE followed by autoradiography. In blood sample of test mice, a protein was also recognized by anti-hGM-CSF antibody using ELISA. The immunohistochemical analysis showed that BmrhGM-CSF was detected within intestinal histiocyte. This indicated it might be absorbed into blood via intestinal microvillus. Pharmacokinetics analysis after orally administered BmrhGM-CSF in animal model of leucopenia including mice, Beagle dogs and macaques showed that: (1) BmrhGM-CSF promoted the CFU-S formation in mice spleen and the synthesis of DNA in bone marrow cells of mice; (2) BmrhGM-CSF induced bone marrow karyocyta granulocyte growth significantly in both macaques and Beagle dogs compared to the negative control group. On the 9th day of orally administration, the animal WBC significantly increased in a dose-dependant manner, in which neutrophilic granulocyte was predominant. The WBC level of dogs in high dose group was about 1.5x10(9)cells/L more than that in the negative control. And the bone marrow smear revealed that the percents of both myloblast and progranulocyte in WBC in the hGM-CSF group were obviously higher than those in the negative control. These results proved that BmrhGM-CSF, a 29kDa glycoprotein expressed by Silkworm pupae bioreactor, could bring into the effect as active cytokine through oral administration.

Administration, Oral↗

Mode of action, purification and amino acid sequence of plantaricin C19, an anti-Listeria bacteriocin produced by Lactobacillus plantarum C19.

Plantaricin C19, an anti-Listeria bacteriocin, was successfully purified by adsorption to and release from producing cells at low pH combined with reverse phase high-performance liquid chromatography (HPLC). The purification resulted in a 900-fold increase in specific activity with a yield of 15% of the original activity. Mass spectrometry analysis gave a molecular weight of 3845.3. Protein microsequencing identified 36 amino acids. Plantaricin C19 is rich in both hydrophobic and basic amino acids in good accordance with its basic and hydrophobic character. Comparison of the amino acid sequence of plantaricin C19, with the sequence of some other anti-Listeria bacteriocins produced with lactic acid bacteria, revealed that plantaricin C19 has in its N-terminal region the consensus sequence--YYGNGL--(uniquely with Valine instead of Leucine as found in all other bacteriocins), identifying plantaricin C19 as a pediocin-like bacteriocin. Plantaricin C19 exerted a bacteriostatic action on sensitive cells of Listeria grayi IP 6818 in BHI broth. No loss of intracellular K+, Mg2+ or UV-absorbing materials was observed. Adsorption of plantaricin C19 on L. grayi CIP 6818 decreased in the presence of salts.

Amino Acid Sequence↗

Detection of Actinobacillus pleuropneumoniae in cultures from nasal and tonsillar swabs of pigs by a PCR assay based on the nucleotide sequence of a dsbE-like gene.

A PCR assay for the detection of Actinobacillus pleuropneumoniae was developed based on the amplification of a dsbE-like gene. All of 157 field isolates of A. pleuropneumoniae reacted in the PCR by the amplification of a 342bp product. No reaction was observed with related bacterial species or other bacterial species isolated from pigs, except for A. lignieresii. The lower detection limit of the PCR was 10(2) CFU per PCR test tube and was not affected by the addition of 10(6) CFU Escherichia coli. The PCR was evaluated on mixed bacterial cultures from nasal and tonsillar swabs as well as suspensions of nasal conchae and tonsils obtained from specific pathogen-free (SPF) pigs, experimentally infected pigs, and pigs from farrow-to-finish herds. The results of the new PCR were compared with a PCR based on the detection of the omlA gene coding for an outer membrane protein, with a commercially available PCR (Adiavet APP, Adiagène, Saint-Brieuc, France), and with conventional culturing. No positive reactions were observed with any of the PCR methods in samples of SPF animals. In samples of the other animals, no or low significant differences between nasal swabs and suspensions as well as tonsillar swabs and suspensions were observed in any method. In general, more positive results were obtained from tonsillar samples in comparison to nasal samples. Interassay sensitivity and specificity values were assessed for each test by pair wise comparisons between assays. The agreement between tests was evaluated by calculating Cohen's kappa coefficient. From these analyses the three PCR assays showed a good agreement. The dsbE-based PCR proved to be highly sensitive (95 and 93%) and specific (82 and 74%) in comparison to the omlA-based PCR and the commercially available PCR, respectively. It was concluded that the dsbE-like gene-based PCR is a reliable diagnostic assay for demonstration of A. pleuropneumoniae. Furthermore, it was demonstrated that tonsillar swabs can be used for the detection of the pathogen in healthy carrier animals.

Actinobacillus Infections↗

Identification of the first isolates of Trichosporon asahii var asahii from disseminated trichosporonosis in China.

Infection with Trichosporon asahii is a major cause of deep-seated and disseminated trichosporonosis, which is associated with a high mortality rate. Disseminated trichosporonosis in individuals with no underlying disease has not been reported. In this study, we report the identification of the first isolate of Trichosporon asahii var. asahii in China. Two isolates were obtained from the liver and skin of a patient with disseminated trichosporonosis who displayed no evidence of underlying disease. The morphologic and physiologic characteristics of the two isolates differed slightly from those of usual strains of T. asahii var. asahii, including the type strain CBS 2479. Ubiquinone-9 was identified as the major ubiquinone in both isolates. Sequence analysis of the LSUrDNA D1/D2, ITS, and IGS1 regions from the two isolates showed them to be T. asahii var. asahii, and random amplified polymorphic DNA analysis strongly suggested that they were the same strain.

Adult↗

Interactions between stem cell factor and c-Kit are required for intestinal immune system homeostasis.

Interactions between stem cell factor (SCF) and its receptor, c-Kit, are important for development of hematopoietic, melanocytes, and germ cells. T lymphocytes appeared normal in c-Kit (W/Wv) or SCF (SI/SId) mutant mice, except for those residing within the intestinal epithelium, the intraepithelial lymphocytes (IEL). Normally, IEL are composed of equal numbers of cells with alpha beta or gamma delta T cell receptors. In mutant mice, beginning at 6-8 weeks of age, the number of gamma delta IEL decreased, whereas alpha beta IEL increased. The latter was due largely to an increased CD4+ CD8+ TCR alpha beta subset, suggesting that these cells may be intermediates in the alpha beta IEL lineage. c-Kit or SCF was expressed by IEL or intestinal epithelial cells, respectively, indicating a potential for direct intercellular interaction. This possibility was supported by reconstitution studies that demonstrated that c-Kit mutations directly affected IEL. Thus, SCF-c-Kit interactions are important for homeostasis of the intestinal immune compartment.

Animals↗

Plasmodium falciparum EBA-175 kDa protein peptides which bind to human red blood cells.

Solid experimental evidence indicates that EBA-175 is used as a ligand by the Plasmodium falciparum merozoite to bind to human RBC, via different binding processing fragments. Using synthetic peptides and specific receptor-ligand interaction methodology, we have identified 6 high-activity binding sequences from the EBA-175 CAMP strain; peptide 1758 (KSYGTPDNIDKNMSLIHKHN), located in the so-called region I for which no binding activity has been reported before, peptides 1779 (NIDRIYDKNLLMIKEHILAI) and 1783 (HRNKKNDKLYRDEWWKVIKK), located in region II, in a sub-region known as 5' Cys F2, previously reported as being a binding region, and peptides 1814 (DRNSNTLHLKDYRNEENERH), 1815 (YTNQNINISQERDLQKHGFH) and 1818 (NNNFNNIPSRYNLYDKKLDL), in region III-V where antibodies inhibit merozoite invasion of erythrocytes. The affinity constants were between 60 and 180 nM and the critical amino acids involved in the binding were identified. The binding of these peptides to enzyme-treated RBC was analysed; binding of peptide 1814, located in the III-V region, was found to be sialic acid dependent. Some of these high binding peptides were able to inhibit in vitro merozoite invasion and to block the binding of recombinant RII-EBA to RBC. Several of these peptides are located in regions recognized by protective immune clusters of merozoites (ICMs) eluted antibodies.

Amino Acid Sequence↗

Tokaracetin, a new platelet antagonist that binds to platelet glycoprotein ib and inhibits von Willebrand factor-dependent shear-induced platelet aggregation.

A new platelet antagonist, tokaracetin, was isolated from the venom of Trimeresurus tokarensis by ion-exchange chromatography, heparin-Sepharose chromatography and hydrophobic HPLC. The purified protein showed an apparent molecular mass on SDS/PAGE of 28.9 kDa under non-reducing conditions. On reduction, 16.1 and 15.4 kDa subunits were observed, suggesting that the molecule is a heterodimer. Tokaracetin inhibited the binding of 125I-labelled bovine von Willebrand factor (vWF) and 125I-labelled human vWF in the presence of botrocetin to fixed human platelets. It did not block ADP-, collagen- or thrombin receptor agonist peptide-induced platelet aggregation in human platelet-rich plasma (PRP), or induce platelet agglutination in PRP. On reduction, tokaracetin lost its inhibitory activity on the agglutination of fixed human platelets by bovine vWF. 125I-Tokaracetin specifically bound to washed human platelets with high affinity (Kd 3.9 +/- 1.4 nM) at 47,440 +/- 2780 binding sites per platelet. Binding of tokaracetin to fixed human platelets was reversible, and was inhibited by monoclonal antibody GUR83-35, which is directed against the N-terminal vWF-binding domain of human glycoprotein Ib (GPIb). Tokaracetin completely inhibited vWF-dependent shear-induced platelet aggregation in PRP at 3 micrograms/ml. The N-terminal amino acid sequences of tokaracetin subunits showed a high degree of identity with those of alboaggregin-B. These results suggest that this new platelet antagonist may be a useful tool in the development of specific inhibitors of the vWF-GPIb interaction.

Amino Acid Sequence↗

Characterization of nickel-resistant bacteria isolated from serpentine soil.

In the present study, heterotrophic nickel-resistant bacteria were isolated and characterized from three different serpentine outcrops in central Italy populated by the nickel-hyperaccumulating plant Alyssum bertolonii. Bacteria were isolated from the rhizosphere of the plant and from soil portions at various distances from the plant. The proportion of nickel-resistant cfu was higher in proximity to the plant than in free soil. A total of 138 isolates was collected and grouped into 47 different operational taxonomic units (OTUs) by means of amplified ribosomal DNA restriction analysis (ARDRA) and into 25 heavy-metal resistant phenotypes. The phylogenetic position of strains belonging to 20 OTUs, representing more than the 70% of the total isolates, was determined by 16S rDNA sequencing. These analyses showed that the most represented genera in all three different outcrops were Pseudomonas and Streptomyces. Pseudomonas strains were found to be predominant in the plant rhizosphere, whereas Streptomyces strains were mainly present in the soil.

Brassicaceae↗

Depletion of Vbeta5.2/5.3 T cells with a humanized antibody in patients with multiple sclerosis.

A potentially pathogenic expansion of T cells expressing T cell receptor (TCR) Vbeta5.2/5.3 has been demonstrated in patients with multiple sclerosis (MS). A humanized antibody (ATM-027) directed against these T cells has been developed to further investigate the role of this subpopulation of T cells in MS. The pharmacokinetics/dynamics and safety of ATM-027 (0.3-300 mg intravenously over 30 min) were investigated in 14 patients with MS. The effect of treatment on cytokine expression and autoreactivity to peptides of myelin basic protein (MBP) was also studied. ATM-027 was well tolerated and raised no safety concerns. Clearance of the antibody was low and elimination half-life was approximately 3 weeks. The majority of the target Vbeta5.2/5.3 expressing T cells were depleted for at least 18 months. The small remaining fraction of target cells showed a marked decrease in their TCR expression, which was recovered within 8 months. The numbers of peripheral blood mononuclear cells (PBMCs) with spontaneous expression of IFN-gamma was decreased at 72 h and 8 weeks after treatment, whilst no clear effects on TNF-alpha, IL-4, IL-10, TGF-beta expression were observed. There was also a significant decrease in the number of PBMCs producing IFN-gamma in response to MBP peptide 80-102. We conclude that long-term depletion of T cells expressing defined Vbeta subgroups in MS patients is feasible using selective immunotherapy. The selective depletion of Vbeta5.2/5.3 expressing T cells in this study resulted in a decrease in potentially disease promoting anti-MBP reactivity and pro-inflammatory cytokine production.

Adolescent↗

Early-onset multifocal inflammation in the transforming growth factor beta 1-null mouse is lymphocyte mediated.

Transforming growth factor beta 1 (TGF beta 1)-null mice die fro complications due to an early-onset multifocal inflammatory disorder. We show here that cardiac cells are hyperproliferative and that intercellular adhesion molecule 1 (ICAM-1) is elevated. To determine which phenotypes are primarily caused by a deficiency in TGF beta 1 from those that are secondary to inflammation, we applied immunosuppressive therapy and genetic combination with the severe combined immunodeficiency (SCID) mutation to inhibit the inflammatory response. Treatment with antibodies to the leukocyte function-associated antigen 1 doubled longevity, reduced inflammation, and delayed heart cell proliferation. TGF beta 1-null SCID mice displayed no inflammation or cardiac cell proliferation, survived to adulthood, and exhibited normal major histocompatibility complex II (MHC II) and ICAM-1 levels. TGF beta 1-null pups born to a TGF beta 1-null SCID mother presented no gross congenital heart defects, indicating that TGF beta 1 alone does not play an essential role in heart development. These results indicate that lymphocytes are essential for the inflammatory response, cardiac cell proliferation, and elevated MHC II and ICAM-1 expression, revealing a vital role for TGF beta 1 in regulating lymphocyte proliferation and activation, which contribute to the maintenance of self tolerance.

Animals↗

Mutations are involved in emergence of aminoglycoside-induced small colony variants of Staphylococcus aureus.

Staphylococcus aureus small colony variants (SCVs) occur frequently after local treatment with aminoglycosides and cause persistent as well as recurrent infections. So far, the molecular mechanism of the emergence of SCVs is not understood and regulatory as well as genetic mechanisms seem conceivable. To screen for possible mutations, the hemin biosynthetic gene cluster of a gentamicin-induced SCV was sequenced and was found to contain a deletion in the gene hemH. To further assess the influence of a high mutation rate on the development of SCVs, we tested the emergence of SCVs in a strain that had been inactivated in the DNA proofreading enzyme MutS. In the mutant, spontaneous SCVs emerged 556-fold more frequently than in the parent strain. By incubation in the presence of subinhibitory concentrations of gentamicin, the SCV frequency in the parent strain could be increased to 9.7 x 10(-6), whereas it remained rather stable in the mutant (1.8 x 10(-5)). Eighty percent of the gentamicin-induced SCVs were hemin auxotrophic in contrast to only 20% of the spontaneous SCVs which may explain the large proportion of hemin auxotrophs among clinical SCVs from patients previously treated with aminoglycosides. Additionally, a clinical S. aureus SCV isolate with a mutator phenotype, indicated by the generation of rifampicin-resistant mutants at a 16-fold higher frequency than in the reference strain S. aureus NCTC 8325, was characterized. The results demonstrate that a high mutation rate favours the emergence of SCVs, and suggest that mutations in general play an important role in the development of SCVs.

Amino Acid Sequence↗

Relaxed negative selection in germinal centers and impaired affinity maturation in bcl-xL transgenic mice.

The role of apoptosis in affinity maturation was investigated by determining the affinity of (4-hydroxy-3-nitrophenyl)acetyl (NP)-specific antibody-forming cells (AFCs) and serum antibody in transgenic mice that overexpress a suppressor of apoptosis, Bcl-xL, in the B cell compartment. Although transgenic animals briefly expressed higher numbers of splenic AFCs after immunization, the bcl-xL transgene did not increase the number or size of germinal centers (GCs), alter the levels of serum antibody, or change the frequency of NP-specific, long-lived AFCs. Nonetheless, the bcl-xL transgene product, in addition to endogenous Bcl-xL, reduced apoptosis in GC B cells and resulted in the expansion of B lymphocytes bearing VDJ rearrangements that are usually rare in primary anti-NP responses. Long-lived AFCs bearing these noncanonical rearrangements were frequent in the bone marrow and secreted immunoglobulin G(1) antibodies with low affinity for NP. The abundance of noncanonical cells lowered the average affinity of long-lived AFCs and serum antibody, demonstrating that Bcl-xL and apoptosis influence clonal selection/maintenance for affinity maturation.

Animals↗

Characterization and evolutionary analysis of a large polygalacturonase gene family in the oomycete plant pathogen Phytophthora cinnamomi.

Polygalacturonases (PGs) are secreted by fungal pathogens during saprophytic and parasitic growth, and their degradation of pectin in the plant cell wall is believed to play a major role in tissue invasion and maceration. In this study, PG activity was demonstrated in culture filtrates of the oomycete plant pathogen, Phytophthora cinnamomi. A P. cinnamomi pg gene fragment amplified using degenerate primers based on conserved regions in fungal and plant PGs was used to isolate 17 complete P. cinnamomi pg genes and pseudogenes from a genomic library and partial sequence for another two genes. Gel blotting of genomic DNA indicated that there may be even more pg genes in the P. cinnamomi genome. P. cinnamomi pg gene sequences were expressed in PG-deficient yeast and found to confer PG activity, thereby confirming their functional identity. The predicted mature P. cinnamomi PGs fall into subgroups that exhibit large differences in the extent of N-glycosylation, isoelectric points, and N- and C-terminal structure. Evidence for birth-and-death and reticulate evolution in the P. cinnamomi pg gene family was obtained, and some codons for surface exposed residues in the P. cinnamomi PGs were shown to have been subject to diversifying selection. Contrary to accepted phylogenies for other proteins, phylogenetic analysis of the P. cinnamomi PGs revealed a closer relationship with PGs from true fungi than with those from plants.

Amino Acid Sequence↗

Fungal colonization of soil-buried plasticized polyvinyl chloride (pPVC) and the impact of incorporated biocides.

Plasticized polyvinyl chloride (pPVC) with or without incorporated biocides was buried in grassland and forest soil for up to 10 months. The change with time in viable counts of fungi on the plastic surface was followed, together with the percentage capable of clearing the two plasticizers dioctyl adipate (DOA) and dioctyl phthalate (DOP). With time fungal total viable counts (TVC) on control pPVC increased and the fraction able to clear DOA was considerably higher than the average estimated in both soil types. A total of 92 fungal morphotypes were isolated from grassland soil and 42 from forest soil with the greatest variety of fungal isolates observed on control pPVC. The incorporation of biocides into pPVC affected both fungal TVC and the richness of species isolated. The biocides NCMP [n-(trichloromethylthio)phthalimide], OBPA (10,10'-oxybisphenoxarsine) and OIT (2-n-octyl-4-isothiazolin-3-one) were the most effective in grassland soil, and TCMP [2,3,5,6-tetrachloro-4-(methylsulphonyl)pyridine] and NCMP the most effective in forest soil. In grassland soil, Penicillium janthinellum established as a principal colonizer and was recovered from all pPVC types. DOP clearers were found at much lower levels than DOA clearers, with Doratomyces spp. being the most efficient. At the end of 10 months the physical properties of the pPVC were altered; changes in stiffness were the most significant for heavily colonized grassland-buried pPVC samples, whereas in forest soil, the extensibility of the pPVC was affected more than the stiffness. These results suggest that fungi are important colonizers of pPVC buried in soil and that enrichment of soil fungi capable of clearing DOA occurs during colonization of the plastic surface. The results also demonstrate that incorporated biocides have a marked impact on the richness of species colonizing the pPVC surface.

Adipates↗

A cell-counting factor regulating structure size in Dictyostelium.

Developing Dictyostelium cells form large aggregation streams that break up into groups of 0.2 x 10(5) to 1 x 10(5) cells. Each group then becomes a fruiting body. smlA cells oversecrete an unknown factor that causes aggregation streams to break up into groups of approximately 5 x 10(3) cells and thus form very small fruiting bodies. We have purified the counting factor and find that it behaves as a complex of polypeptides with an effective molecular mass of 450 kD. One of the polypeptides is a 40-kD hydrophilic protein we have named counting. In transformants with a disrupted counting gene, there is no detectable secretion of counting factor, and the aggregation streams do not break up, resulting in huge (up to 2 x 10(5) cell) fruiting bodies.

Amino Acid Sequence↗

The effects of transgalactosylated oligosaccharides on gut flora metabolism in rats associated with a human faecal microflora.

The effect on various caecal bacteria and their metabolic activities of feeding diet containing transgalactosylated oligosaccharides (TOS) with or without Bifidobacterium breve (administered in the drinking water) was investigated in rats colonized with a human faecal microflora. TOS (5% w/w in diet) or TOS plus B. breve, given for 4 weeks, induced increases in caecal concentration of total anaerobic bacteria, lactobacilli and bifidobacteria, and decreases in numbers of enterobacteria. Caecal pH was significantly reduced by feeding TOS, as were the activities of beta-glucuronidase and nitrate reductase. In contrast, beta-glucosidase activity was increased in TOS-fed rats. Dietary TOS was also associated with decreased conversion, by caecal contents, of the dietary carcinogen 2-amino-3-methyl-3H-imidazo[4,5-f] quinoline (IQ) to its genotoxic 7-hydroxy derivative.

Animals↗

Investigation of the role of sterol delta 8-->7-isomerase in the sensitivity of Saccharomyces cerevisiae to fenpropimorph.

Treatment of Saccharomyces cerevisiae with the morpholine fungicide fenpropimorph was examined using both a wild-type and a mutant strain (erg2) defective in sterol delta 8-->7-isomerase. No resistance to fenpropimorph was observed in the mutant strain after 3 days, although after 7 days the mutant and the wild-type strains had grown in concentrations of fenpropimorph close to the saturating dose. Re-inoculation of both strains into fresh medium containing fenpropimorph resulted in continued growth and this adaptation to fungicide tolerance was lost on subculture in the absence of fenpropimorph. Analysis of the sterols present in the cells indicated that fenpropimorph treatment resulted in the accumulation of delta 8,14-sterols. This accumulation and the corresponding depletion of ergosterol were correlated with growth inhibition rather than the presence of delta 8-sterols. Together with an absence of gene dosage effect for ERG2 on fenpropimorph sensitivity, this supports the hypothesis that sterol delta 8-->7-isomerase inhibition does not contribute to the fungicidal activity of fenpropimorph.

Base Sequence↗