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PubMed1997

Brain and erythrocyte anion transporter protein, band 3, as a marker for Alzheimer's disease: structural changes detected by electron microscopy, phosphorylation, and antibodies.

Band 3, a ubiquitous membrane transport, regulatory, and structural protein, is represented in brain by at least 4 isoforms. Bands 3 in brain performs the same functions as it does in erythrocytes (RBC). It is susceptible to oxidative damage, which, ultimately, terminates its life and that of the cell. We examined the changes band 3 undergoes in Alzheimer's disease (AD) because our previous studies suggest that band 3 is a pivotal protein in neurological disease. Because we hypothesize that AD is a total body disease, we examined peripheral blood cells as well as brain tissue to determine whether the same changes occur in both. Our results indicate that posttranslational changes occur in RBC band 3 that parallel changes in brain band 3. These include decreased 32P-phosphate labeling in vitro of band 3 polypeptides in brain and RBC, increased degradation of band 3, alteration in band 3 recognized by polyclonal and monoclonal antibodies, and decreased anion and glucose transport by blood cells. Serum autoantibodies to band 3 peptides 588-602 and 822-839 were increased in AD patients compared to controls. These band 3 residues lie in anion transport/binding regions. This is consistent with alteration of this region in AD since it is recognized as antigenically different by the patients' immune system. Our data support an immunological component to AD. The finding that changes in RBC in AD reflect those in brain and can be recognized by antibodies should facilitate development of blood tests for diagnosis and monitoring, and early therapy. It is anticipated that identification of molecular sites of posttranslational modification of band 3 will enable us to design specific preventive and treatment strategies, and target drugs to crucial molecular sites.

Aging↗

PubMed2002

Oxygen-limited autotrophic nitrification/denitrification by ammonia oxidisers enables upward motion towards more favourable conditions.

The hypothesis is formulated that in case of oxygen limitation in the sediment, nitrifiers switch from nitrification to oxygen-limited autotrophic nitrification-denitrification (OLAND) in order to survive and maintain activity. During OLAND, ammonium is oxidised using nitrite as e-acceptor to form dinitrogen gas. As an additional advantage they benefit from the gaseous N(2) formed as a means of transport. In this way, the nitrifiers can move out of the sediment and rise through the water column towards more favourable conditions. At the surface, the bacteria could take up oxygen, and recommence nitrification. In order to test this hypothesis, nitrifying sediment with an overlaying water column was simulated in lab-scale columns. Nitrogen transformations and material transport through the water column were followed after addition of different forms of nitrogen under oxygen-limited conditions. (15)N-labelling experiments showed a large contribution of OLAND to the observed nitrogen deficits. Nitrifier enumerations, fluorescent in situ hybridisation and 16S rRNA gene analysis revealed increased populations of ammonia oxidising nitrifiers in the upper water layers. The results presented support the proposed hypothesis of transport using OLAND. Nitrifying activity in the sediment immediately recovered almost completely from prolonged oxygen-limited incubation when oxygen concentrations were increased.

Anaerobiosis↗

PubMed2005

Glomus intraradices dominates arbuscular mycorrhizal communities in a heavy textured agricultural soil.

Arbuscular mycorrhizal fungal (AMF) spore communities were surveyed in a long-term field fertilization experiment in Switzerland, where different amounts of phosphorus (P) were applied to soil. Plots receiving no P as well as plots systematically fertilized in excess to plant needs for 31 years were used to test the hypothesis that application of P fertilizer changes the composition and diversity of AMF communities. AMF spores were isolated from the field soil, identified, and counted so as to quantify the effect of P fertilization on AMF spore density, composition, and diversity. Trap cultures were established from field soil with four host plants (sunflower, leek, maize, and Crotalaria grahamiana), and the spore communities were then analyzed in substrate samples from the pots. Altogether, nine AMF species were detected in the soil. No evidence has been acquired for effect of P fertilization on spore density, composition, and diversity of AMF in both the field soil and in trap cultures. On the other hand, we observed strong effect of crop plant species on spore densities in the soil, the values being lowest under rapeseed and highest under Phacelia tanacetifolia covercrop. The identity of plant species in trap pots also significantly affected composition and diversity of associated AMF communities, probably due to preferential establishment of symbiosis between certain plant and AMF species. AMF spore communities under mycorrhizal host plants (wheat and Phacelia in the fields and four host plant species in trap pots) were dominated by a single AMF species, Glomus intraradices. This resulted in exceptionally low AMF spore diversity that seems to be linked to high clay content of the soil.

Biodiversity↗

PubMed1993

Redistribution of epidermal growth factor receptor as a function of cell density, cell-cell adhesion and calcium in human (A-431) cells.

Cell surface expression of the epidermal growth factor (EGF) receptor in several cell lines declines as a function of increased cell density and is associated with diminished responsiveness to EGF. However, the mechanism whereby this density-induced down regulation of receptors occurs has not been discerned. In the present study the distribution of the EGF receptor in A-431 cells as a function of cell density using (1) two polyclonal antibodies raised against peptide specific sequences of the EGF receptor that recognize either the cytoplasmic or extracellular domains of the receptor, respectively, and (2) biotinylated EGF, a specific probe for the cell surface receptor is now investigated. Immunolocalization of the receptor using the polyclonal antibodies or the biotin-EGF revealed that the receptor was homogeneously distributed on the cell surface of individual cells, or in cells plated at low density. In contrast, as cell density increased, prominent EGF immunoreactivity and biotin-EGF staining became limited to the periphery of the cells, at sites of cell-cell apposition, and was characterized by a honeycomb pattern, typical of a basolateral distribution. The effects of low Ca++ treatment, known to cause cells to round up and detach from one another, on EGF receptor distribution in cells at high cell density were then examined. Confocal microscopy of immunostained preparations revealed that incubation of high density cultures in Ca(++)-free media for as little as 10 min restored the homogeneous distribution of the EGF receptor and resulted in strong intracellular staining. Three-dimensional reconstruction of serial optical sections revealed that redistribution of the EGF receptor following low Ca++ treatment involved a heretofore undetected 'ruffling', an immunostaining pattern characterized by stripes of intense fluorescence signal interspersed with complete absence of fluorescence. Next, cell-cell adhesion was disrupted with antisera to the cell adhesion molecule E-cadherin. Although the antisera caused cells to detach from one another, eventually leading to cell rounding and redistribution of the EGF receptor, the receptor 'ruffling' immunostaining pattern rendered by the low Ca++ treatment was not detected. These results suggest that an association may exist between the plasma membrane EGF receptor distribution, density-induced EGF receptor down regulation, and the growth effects of low Ca++ observed in previous studies.

Amino Acid Sequence↗

PubMed2006

Dual function of fish hepcidin: response to experimental iron overload and bacterial infection in sea bass (Dicentrarchus labrax).

The role of hepcidin in iron metabolism regulation and bacterial infection has been the focus of recent attention. However, in spite of the growing number of hepcidin genes known from different organisms, little is known about its putative dual function in fish. The aim of this study was to characterize the sea bass hepcidin gene and to study its role in iron metabolism and infection. The novel sea bass hepcidin gene was found to be organized into two introns and three exons with several copies present in the genome. The transcript showed a constitutive low basal expression being mainly expressed in liver and encoding a putative 85 residues long peptide. Fish were submitted either to iron status modulation or bacterial infection and the hepcidin transcript levels were analysed along with a number of other parameters. Liver hepcidin expression was found to increase in both the iron-overloaded and infected fish, while in the iron-deficient fish no alteration in expression levels was detected. These results point to the evolutionary conservation of hepcidin's dual functions.

Amino Acid Sequence↗

PubMed2004

A von Willebrand factor-binding protein from Staphylococcus lugdunensis.

In the present study, a phage display library covering the genome of Staphylococcus lugdunensis, was affinity-selected against von Willebrand factor (vWf). This led to the identification of a gene, vwbl, encoding a putative cell surface protein of 2060 amino acids, denoted vWbl. The deduced protein has an overall organisation typical of staphylococcal cell surface proteins, with an N-terminal signal peptide, and a C-terminal cell wall sorting signal. The vWf-binding part is located in repetitive domains and antibodies against vWbl or vWf can inhibit the binding. Southern blot analysis showed that vwbl was present in the 12 S. lugdunensis strains tested.

Amino Acid Sequence↗

PubMed2006

Transcarboxylase mRNA: a marker which evidences P. freudenreichii survival and metabolic activity during its transit in the human gut.

Dairy propionibacteria have recently been considered as probiotics which may beneficially modulate the intestinal ecosystem. However, appropriate vectors (food matrices containing the probiotic) which preserve their viability and offer good tolerance towards digestive stresses need to be developed. In addition, the development of efficient non-invasive methods which specifically monitor Propionibacterium freudenreichii concentration and activity within the human gut is required. To address this latter need, an enzyme involved in propionic fermentation, transcarboxylase, was evaluated in this study as molecular marker in P. freudenreichii. In vitro, the three transcarboxylase subunits were shown to be encoded by an operon and their expression regulated. It occurred during propionic fermentation, ceased in starved cells and was not affected by digestive stresses. The 5S subunit gene of transcarboxylase allowed specific detection of P. freudenreichii by real time PCR in the complex human faecal microbiota. A dairy vector harbouring P. freudenreichii was developed and afforded elevated probiotic faecal concentrations in humans. In vivo, this PCR method allowed rapid quantification of faecal P. freudenreichii in agreement with the cultural method (cfu counting). Moreover, real time Reverse Transcription (RT) -PCR evidenced transcription of the 5S subunit gene during transit through the human digestive tract. This work constitutes a methodological advance for survival and activity evaluation in human trials of the probiotics belonging to the P. freudenreichii species. It strongly suggests that this bacterium not only survives but remains metabolically active in the human gut.

Base Sequence↗

PubMed2004

Subfamily I Treponema pallidum repeat protein family: sequence variation and immunity.

A 12-membered Treponema pallidum repeat (Tpr) protein family has been identified in T. pallidum subsp. pallidum, the causative agent of syphilis. The subfamily I Tpr proteins (C, D, F, and I) possess conserved sequence at the N- and C-termini and central regions that differentiate the members. These proteins may be important in the immune response during syphilis infection and in protective immunity. Strong antibody responses have been observed toward some of the subfamily I Tpr proteins during infection with different syphilis isolates. Some sequence variation has also been identified in one subfamily I Tpr member, TprD, among T. pallidum subsp. pallidum isolates. In this study, we examined sequences in the remaining subfamily I Tpr proteins among strains. Both TprF and TprI were conserved among T. pallidum subsp. pallidum isolates. While some heterogeneity was identified in TprC. We further examined the immune response and protective capacity of TprF protein in this paper. We demonstrate that the N-terminal conserved region of the subfamily I Tpr proteins elicits strong antibody and T-cell responses during infection, and immunization with this region attenuates syphilitic lesion development upon infectious challenge.

Amino Acid Sequence↗

PubMed2006

Impacts of 2,4-D application on soil microbial community structure and on populations associated with 2,4-D degradation.

The effect of 2,4-dichlorophenoxyacetic acid (2,4-D) application rate on microbial community structure and on the diversity of dominant 2,4-D degrading bacteria in an agricultural soil was examined using cultivation-independent molecular techniques coupled with traditional isolation and enumeration methods. Fingerprints of microbial communities established under increasing concentrations of 2,4-D (0-500 mg kg-1) in batch soil microcosms were obtained using denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA gene segments. While a 2,4-D concentration of at least 100 mg kg-1 was required to obtain an apparent change in the community structure as visualized by DGGE, the greatest impact of 2,4-D concentration occurred in the 500 mg kg-1 treatment, resulting in significantly reduced diversity of the dominant populations and enrichment by Burkholderia-like populations. The greatest diversity of 2,4-D degrading isolates was cultivated from the 10 mg kg-1 treatment, indicating that under these conditions, cultivation was more sensitive than DGGE for detecting changes in community structure. Most of these isolates harbored homologs of Ralstonia eutrophus JMP134 and Burkholderia cepacia tfdA catabolic genes. Results from this study revealed that agriculturally relevant application rates of 2,4-D may provide a temporary selective advantage for organisms capable of utilizing 2,4-D as a carbon and energy source.

2,4-Dichlorophenoxyacetic Acid↗

PubMed2000

Quantitative and qualitative analyses of the immune responses induced by a multivalent minigene DNA vaccine.

Vaccines containing minigenes - isolated antigenic epitopes encoded by short open reading frames - can, under certain circumstances, confer protective immunity upon the vaccinee. Here we evaluate the efficacy of the minigene vaccine approach using DNA immunization and find that, to be immunogenic, a minigene-encoded epitope requires a perfect "Kozak" translational initiation region. In addition, using intracellular cytokine staining, we show that immunization with a plasmid encoding a full-length protein induces epitope-specific CD8(+) T cells which are detectable directly ex vivo, and constitute approximately 2% of the vaccinee's splenic CD8(+) T cells. In contrast, such cells are undetectable directly ex vivo in recipients of a minigene vaccine. Nevertheless, the minigene plasmid does induce a low number of epitope-specific CD8(+) T cells, which can be amplified to detectable levels by in vivo stimulation. Indeed, 4 days after in vivo stimulation (by virus infection), all vaccinated mice - regardless of whether they had been vaccinated with the minigene or with the full-length gene - had similar numbers of epitope-specific CD8(+) T cells. However, despite these strong responses at 4 days post-infection, recipients of the minigene vaccine showed no enhanced ability to limit virus replication and dissemination. We therefore observe a dichotomy; minigene vaccinees are not protected, despite the presence of strong virus-specific immune responses at 4 days post-challenge. We suggest that the protective benefits of vaccination exert themselves very soon - perhaps within minutes or hours - after virus challenge. If the vaccine-induced immune response is too low to achieve this early protective effect, virus-specific T cells will expand rapidly, but ineffectually, leading to the strong but non-protective response measured at 4 days post-infection. Thus, vaccine-induced immunity should be monitored very early in infection, since the extent to which these responses may later be amplified is largely irrelevant to the protection observed.

Amino Acid Sequence↗

PubMed2004

Streptococcus alactolyticus is the dominating culturable lactic acid bacterium species in canine jejunum and feces of four fistulated dogs.

Canine intestinal lactic acid bacterium (LAB) population in four fistulated dogs was cultured and enumerated using MRS agar. LAB levels ranging from 1.4x10(6) to 1.5x10(7) CFU ml(-1) were obtained in jejunal chyme. In the fecal samples 7.0x10(7) and 2.0x10(8) CFU g(-1) were detected. Thirty randomly selected isolates growing in the highest sample dilutions were identified to species level using numerical analysis of 16S and 23S rDNA restriction fragment length polymorphism patterns (ribotyping) and 16S rDNA sequence analysis. According to these results, Streptococcus alactolyticus was the dominant culturable LAB species in both feces and jejunal chyme. In addition, Lactobacillus murinus and Lactobacillus reuteri were detected.

Animals↗

PubMed2002

Correlation of the antibacterial activities of cationic peptide antibiotics and cationic steroid antibiotics.

The antibacterial activities of cationic steroid antibiotics and cationic peptide antibiotics have been compared. Depolarization of bacterial membranes, activation of bacterial stress-related gene promoters, and changes in bacterial morphologies caused by these antibiotics suggest that cationic steroid and peptide antibiotics share mechanistic aspects. Modified cationic steroid antibiotics display improved selectivity for prokaryotic cells over eukaryotic cells presumably due to increased charge recognition.

Amino Acid Sequence↗

PubMed2003

Indigenous and contaminant microbes in ultradeep mines.

Rock, air and service water samples were collected for microbial analyses from 3.2 kilometres depth in a working Au mine in the Witwatersrand basin, South Africa. The approximately metre-wide mined zone was comprised of a carbonaceous, quartz, sulphide, uraninite and Au bearing layer, called the Carbon Leader, sandwiched by quartzite and conglomerate. The microbial community in the service water was dominated by mesophilic aerobic and anaerobic, alpha-, beta- and gamma-Proteobacteria with a total biomass concentration approximately 10(4) cells ml(-1), whereas, that of the mine air was dominated by members of the Chlorobi and Bacteroidetes groups and a fungal component. The microorganisms in the Carbon Leader were predominantly mesophilic, aerobic heterotrophic, nitrate reducing and methylotrophic, beta- and gamma-Proteobacteria that were more closely related to service water microorganisms than to air microbes. Rhodamine WT dye and fluorescent microspheres employed as contaminant tracers, however, indicated that service water contamination of most of the rock samples was < 0.01% during acquisition. The microbial contaminants most likely originated from the service water, infiltrated the low permeability rock through and accumulated within mining-induced fractures where they survived for several days before being mined. Combined PLFA and terminal restriction fragment length profile (T-RFLP) analyses suggest that the maximum concentration of indigenous microorganisms in the Carbon Leader was < 10(2) cells g(-1). PLFA, 35S autoradiography and enrichments suggest that the adjacent quartzite was less contaminated and contained approximately 10(3) cells gram(-1) of thermophilic, sulphate reducing bacteria, SRB, some of which are delta-Proteobacteria. Pore water and rock geochemical analyses suggest that these SRB's may have been sustained by sulphate diffusing from the adjacent U-rich, Carbon Leader where it was formed by radiolysis of sulphide.

Air Microbiology↗

PubMed2001

The potential of a polyphasic PCR-dGGE approach in evaluating microbial diversity of natural whey cultures for water-buffalo Mozzarella cheese production: bias of culture-dependent and culture-independent analyses.

A polyphasic PCR-DGGE approach was used to describe the microbial population occurring in natural whey cultures (NWCs) for water-buffalo Mozzarella cheese production. Total microbial community was assessed without cultivation by analyzing DNA directly extracted from the original samples of NWC. In addition, DNA extracted from bulks of cells formed by harvesting colonies from the serial dilution agar plates of a variety of culture media was used to profile the "cultivable" community. The 16S rDNA V3 region was amplified using DNA from NWC as well as DNA from bulks as templates and the amplicons were separated by DGGE. The microbial entities occurring in NWCs were identified by partial 16S rDNA sequencing of DGGE bands: four lactic acid bacteria (LAB) closest relative of Streptococcus thermophilus, Lactococcus lactis, Lactobacillus delbrueckii and Lactobacillus crispatus were revealed by the analysis of DNA directly extracted from NWC while two other LAB, Lactobacillus fermentum and Enterococcus faecalis, were identified by analyzing DNA from the cultivable community. The developed PCR-DGGE analysis of the "cultivable" community showed good potential in evaluating microbial diversity of a dairy environment: it usefully highlighted the bias introduced by selective amplification when compared to the analysis of the total community from NWC and allowed suitability of media and growth conditions to be evaluated. Moreover, it could be used to complete the culture independent study of microbial diversity to give information on concentration ratios among species occurring in a particular environment and can be proposed for rapid identification of dominant microorganisms in alternative to traditional tools.

Animals↗

PubMed1995

Comparison of antigen detection, polymerase chain reaction and culture for detection of Chlamydia trachomatis in genital infection.

An in-house polymerase chain reaction (PCR) test using ribosomal RNA gene primers was compared with chlamydia antigen detection (DIF) and culture for the detection of Chlamydia trachomatis. Five hundred and forty-eight fresh (unstored) genital swabs and 174 urines (collected at the same time) from patients attending a sexually-transmitted diseases clinic were examined. PCR, DIF and culture detected chlamydia in 43, 35 and 42 swabs respectively from the 43 resolved positive cases. The specificity on the resolved negative specimens was 100% for each of the tests. From the urines, PCR and DIF detected the organism in 16 and 15 cases respectively of the 23 resolved positive males tested but in only 2 and 3 cases respectively of the 9 resolved positive females tested. Specificities were 100% in all cases. Both of the non-culture tests manifested problems with urine due to inhibitory activity (in PCR test) or excessive debris (in DIF test) in about 5% of the specimens. Culture of the urines yielded sensitivities of 40% in the males and 22% in the females. Overall PCR was more sensitive than either culture or DIF on both urethral and cervical swabs and urines. The urines yielded less than three-quarters the number of positives that was obtained from the swabs and were considered to be an unsatisfactory specimen for chlamydial diagnosis. It is concluded that PCR is a satisfactory alternative to culture on genital swabs and may be preferable in situations where the viability of the organisms is in question. DIF remains useful because of its speed and simplicity but is insufficiently sensitive to be relied upon by itself.

Adolescent↗

PubMed2002

T69D/N pol mutation, human immunodeficiency virus type 1 RNA levels, and syncytium-inducing phenotype are associated with CD4 cell depletion during didanosine therapy.

The contribution of virologic and host factors to CD4 cell depletion associated with human immunodeficiency virus (HIV) type 1 was evaluated in children drawn from a larger efficacy trial of 2 doses of didanosine (ddI) monotherapy (Pediatric AIDS Clinical Trials Group 144). Thirty children, half with stable CD4 cell counts (non-progressors) and half with a marked decline in CD4 cells (progressors), were studied during 60-72 weeks of ddI therapy. The children were matched for age and CD4 cell counts at study entry. Three viral parameters, syncytium-inducing phenotype, higher virus load, and mutation in HIV-1 pol encoding the T69D/N mutation, were associated with disease progression. Disease progression was not associated with mutations in the reverse-transcriptase gene previously associated with resistance to ddI (L74V, K65R, or M184V). The selection of the T69D/N mutation in children with HIV-1 disease progression during ddI therapy suggests that this mutation confers a fitness advantage to the virus that may include resistance to ddI.

Acquired Immunodeficiency Syndrome↗

PubMed2006

Different rates of disease progression of HIV type 1 infection in Tanzania based on infecting subtype.

BACKGROUND: Many different subtypes of human immunodeficiency virus (HIV) type 1 have been identified, particularly in sub-Saharan Africa. However, much remains unknown regarding the relative pathogenicity of these subtypes and their influence on the clinical progression of HIV infection. We examined prospectively the associations between HIV-1 subtypes A, C, and D and recombinant viruses, as well as the rates of disease progression in a cohort of seropositive women from Dar es Salaam, Tanzania. METHODS: A total of 428 pregnant mothers participating in a larger controlled trial of the effect of vitamin supplements were selected for DNA sequencing of their HIV-1 subtype. Plasma viral load was measured at baseline, and CD4+ cell counts was assessed at baseline and at regular intervals throughout the follow-up period. Proportional hazards regression (hazards ratio [HR]) analysis was used to measure the association between viral subtype and the rate of disease progression. RESULTS: Relative to patients with subtype A, patients with subtype D experienced the most rapid progression to death (HR, 2.27; 95% confidence interval [CI], 1.46-3.52) or to the World Health Organization stage 4 of illness (HR, 1.94; 95% CI, 1.20-3.14) and to a CD4+ cell count of <200 cells/mm3 (HR, 2.12; 95% CI, 1.42-3.17). After adjustment for viral load, CD4+ cell count, and other baseline covariates, the associations remained similar. CONCLUSIONS: We observed heterogeneity in the rates of disease progression of HIV-1 disease in infected persons, on the basis of the infecting subtype. Subtype D was associated with the most rapid progression of the disease, relative to the other 3 categories of viruses in our cohort.

Adult↗

PubMed1995

In vivo administration of CD4-specific monoclonal antibody: effect on provirus load in rhesus monkeys chronically infected with the simian immunodeficiency virus of macaques.

Since monoclonal antibodies (MAb) specific for CD4 are potent inhibitors of HIV and SIV replication in vitro, we explored their potential usefulness in vivo as an AIDS therapy. The anti-CD4 MAb 5A8 binds to domain 2 of the CD4 molecule and inhibits virus replication and virus-induced cell fusion at a postvirus binding step. Administration of this MAb to normal rhesus monkeys coats all circulating and lymph node CD4 cells and induces neither CD4 cell clearance nor measurable immunosuppression. In the present study, monkeys chronically infected with the simian immunodeficiency virus of macaques (SIVmac) had stable levels of SIVmac provirus in PBMC prior to treatment as measured by a quantitative polymerase chain reaction technique. Six infected monkeys treated with anti-CD4 MAb demonstrated a significant decrease in SIVmac provirus level after 9 days. Of these monkeys, 3 had > 800 CD4 cells/microliter and developed strong antimouse Ig responses that prevented further treatment. The remaining 3 monkeys had < 800 CD4 cell/microliter and failed to develop antimouse Ig antibody responses. When treatment was continued for 12-21 days in these monkeys, a sustained or further decrease in SIVmac provirus load occurred over the extended treatment period. Four monkeys that received a control MAb of irrelevant specificity for 9-22 days showed either no significant change or a transient increase in SIVmac provirus. Thus, the passive administration of anti-CD4 MAb may exert a specific antiviral effect in controlling immunodeficiency virus infection in vivo.

Animals↗