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PubMed1994

Disruption of the neurotrophin-3 receptor gene trkC eliminates la muscle afferents and results in abnormal movements.

The trkC gene is expressed throughout the mammalian nervous system and encodes a series of tyrosine protein kinase isoforms that serve as receptors for neurotrophin-3 (NT3), a member of the nerve growth factor (NGF) family of neurotrophic factors. One of these isoforms, gp145trkC/TrkC K1, mediates the trophic properties of NT3 in cultured cells. Here we show that homozygous mice defective for TrkC tyrosine protein kinase receptors lack Ia muscle afferent projections to spinal motor neurons and have fewer large myelinated axons in the dorsal root and posterior columns of the spinal cord. These mice display abnormal movements and postures, indicating that NT3/TrkC-dependent sensor; neurons may play a primary role in proprioception, the sense of position and movement of the limbs.

Amino Acid Sequence↗

PubMed2000

Altered expression of expansin modulates leaf growth and pedicel abscission in Arabidopsis thaliana.

Expansins are cell-wall-loosening proteins that induce stress relaxation and extension of plant cell walls. To evaluate their hypothesized role in cell growth, we genetically manipulated expansin gene expression in Arabidopsis thaliana and assessed the consequent changes in growth and cell-wall properties. Various combinations of promoters were used to drive antisense and sense sequences of AtEXP10, which is maximally expressed in the growing leaf and at the base of the pedicel. Compared with controls, antisense lines had smaller rosettes because of shorter petioles and leaf blades and often acquired a twisted leaf morphology. Petiole cells from antisense plants were smaller than controls and their cell walls were significantly less extensible in vitro. Sense plants had slightly longer petioles, larger leaf blades, and larger cells than controls. Abscission at the base of the pedicel, where AtEXP10 is endogenously expressed, was enhanced in sense plants but reduced in antisense lines. These results support the concept that expansins function endogenously as cell-wall-loosening agents and indicate that expansins have versatile developmental roles that include control of organ size, morphology, and abscission.

Arabidopsis↗

PubMed2006

Retrocyclins kill bacilli and germinating spores of Bacillus anthracis and inactivate anthrax lethal toxin.

Theta-defensins are cyclic octadecapeptides encoded by the modified alpha-defensin genes of certain nonhuman primates. The recent demonstration that human alpha-defensins could prevent deleterious effects of anthrax lethal toxin in vitro and in vivo led us to examine the effects of theta-defensins on Bacillus anthracis (Sterne). We tested rhesus theta-defensins 1-3, retrocyclins 1-3, and several analogues of RC-1. Low concentrations of theta-defensins not only killed vegetative cells of B. anthracis (Sterne) and rendered their germinating spores nonviable, they also inactivated the enzymatic activity of anthrax lethal factor and protected murine RAW-264.7 cells from lethal toxin, a mixture of lethal factor and protective antigen. Structure-function studies indicated that the cyclic backbone, intramolecular tri-disulfide ladder, and arginine residues of theta-defensins contributed substantially to these protective effects. Surface plasmon resonance studies showed that retrocyclins bound the lethal factor rapidly and with high affinity. Retrocyclin-mediated inhibition of the enzymatic activity of lethal factor increased substantially if the enzyme and peptide were preincubated before substrate was added. The temporal discrepancy between the rapidity of binding and the slowly progressive extent of lethal factor inhibition suggest that post-binding events, perhaps in situ oligomerization, contribute to the antitoxic properties of retrocyclins. Overall, these findings suggest that theta-defensins provide molecular templates that could be used to create novel agents effective against B. anthracis and its toxins.

Amino Acid Sequence↗

PubMed2004

Common patterns of B cell perturbation and expanded V4-34 immunoglobulin gene usage in autoimmunity and infection.

Features of the lymphocyte population in systemic lupus erythematosus (SLE) include a disordered B cell profile and production of autoantibodies. An additional distinctive perturbation is the overexpression of V4-34-encoded serum immunoglobulins (Ig). A similar rise in V4-34-encoded Ig occurs in normal subjects following infection with certain herpesviruses, and is found in Epstein-Barr virus (EBV)-associated infectious mononucleosis (IM). To assess common and distinctive features of B cells in patients with SLE and IM, we compared the B cell profile and V4-34 gene involvement in patients with SLE and IM. B cell profiles from patients with IM paralleled those of patients with SLE, showing a differential loss of naïve and memory B cells and the maintenance of plasmablast/early plasma cells. Class-switched V4-34-encoded IgG from plasmablast/early plasma cells was evident both in patients with SLE and IM and revealed common features of oligoclonal expansions with most having undergone somatic hypermutation. It has been proposed that, in healthy individuals, expression of the V4-34 gene is specifically censored prior to isotype switch as a control on autoreactivity. If so, censoring is bypassed following EBV infection, after which equilibrium is restored. Continuing high serum levels in SLE may arise either by disordered regulation, or by subclinical reactivation of endogenous virus.

Amino Acid Sequence↗

PubMed1996

Physiological regulation of early and late stages of megakaryocytopoiesis by thrombopoietin.

Thrombopoietin (TPO) has recently been cloned and shown to regulate megakaryocyte and platelet production by activating the cytokine receptor c-mpl. To determine whether TPO is the only ligand for c-mpl and the major regulator of megakaryocytopoiesis, TPO deficient mice were generated by gene targeting. TPO-/- mice have a >80% decrease in their platelets and megakaryocytes but have normal levels of all the other hematopoietic cell types. A gene dosage effect observed in heterozygous mice suggests that the TPO gene is constitutively expressed and that the circulating TPO level is directly regulated by the platelet mass. Bone marrow from TPO-/- mice have decreased numbers of megakaryocyte-committed progenitors as well as lower ploidy in the megakaryocytes that are present. These results demonstrate that TPO alone is the major physiological regulator of both proliferation and differentiation of hematopoietic progenitor cells into mature megakaryocytes but that TPO is not critical to the final step of platelet production.

Animals↗

PubMed2004

Problems in isolation of Campylobacter jejuni from frozen-stored raw milk and bovine fecal samples: genetic confirmation using multiplex PCR.

The objectives of this study were to evaluate the use of various protocols for the isolation of Campylobacter jejuni from bulk tank milk and bovine fecal samples that were stored frozen for varying times, and to develop a rapid DNA-based protocol that distinguishes C. jejuni from other thermophilic Campylobacter spp. The pathogen was recovered from fecal samples that had been stored for 96-251 days at -20 degrees C with glycerol as the cryopreservative. In a separate study, C. jejuni-positive bovine fecal samples were stored at 5 degrees C for up to 70 days without compromising subsequent recovery of the pathogen. However, the pathogen could not be recovered from pathogenpositive fecal samples stored with or without glycerol (5 mL/11 g sample) for 21 days at -20 degrees C. Bolton broth (BB) and Bolton broth with 5% blood (BBB), containing BB supplement, were used for enrichment. Bacterial isolation was achieved by streaking from BB and BBB, and filtering from BBB onto blood-free charcoal cefoperazone deoxycholate agar (CCDA). The use of BB for the recovery of Campylobacter was more sensitive than BBB, and streaking achieved better isolation rates than filtration. Multiplex PCR incorporating thermophilic Campylobacter-specific 23S rRNA and C. jejuni-specific hippuricase gene sequences was used to confirm C. jejuni. All 265 bulk tank milk samples analyzed were negative for C. jejuni, whereas five of 411 (1.2%) fecal samples tested positive. This is the first report that has used a combination of sequences of the two genes in a multiplex format to identify C. jejuni to the species level. The method described has potential for routine use in the detection of thermophilic Campylobacter in farm environmental samples as well as other samples.

Animals↗

PubMed1995

Quantitation of human cytomegalovirus DNA from peripheral blood cells of human immunodeficiency virus-infected patients could predict cytomegalovirus retinitis.

Human cytomegalovirus (CMV) DNA copy number in white blood cells from both human immunodeficiency virus (HIV)-seronegative and HIV-seropositive patients was amplified from the immediate-early region of CMV DNA and quantified by colorimetric detection of the hybridization of the amplification product to a detector oligonucleotide probe in microtiter wells. By Mann-Whitney U test, significantly higher (P < .05, two-tailed) copy numbers of CMV DNA were detected in HIV-seropositive patients with retinitis than in either patients with < 100 CD4 cells/mm3 and no symptomatic CMV disease or HIV-seropositive patients with > 100 CD4 cells/mm3. By prospective monitoring for increases in CMV DNA copy number, it may be possible to identify HIV-seropositive patients who are at imminent risk for development of symptomatic CMV retinitis.

AIDS-Related Opportunistic Infections↗

PubMed1995

Quantitative relationship of circulating p24 antigen with human immunodeficiency virus (HIV) RNA and specific antibody in HIV-infected subjects receiving antiretroviral therapy. The RV43 Study Group.

To better understand the biologic meaning and potential clinical utility of p24 antigen measurements in human immunodeficiency virus (HIV) infection, p24 antigen and antibody and HIV RNA were quantitated in parallel. Specimens (n = 311) were analyzed from 74 participants in a zidovudine treatment study. Parallel antigen and RNA measurements revealed the frequent occurrence of two types of discordant results. First, p24 antigen was often not detected in samples with high antibody levels even when > 10(6) RNA copies/mL were present. Second, in specimens in which p24 antigen was detected, the concentration was greater than expected on the basis of HIV RNA values. These results suggest that optimal use of serum p24 antigen values will require consideration of both specific antibody levels and non-virion associated antigen.

Adult↗

PubMed1995

Resolution of spontaneous bleeding events but failure of pregnancy in fibrinogen-deficient mice.

To explore the role of the key coagulation factor, fibrinogen, in development, hemostasis, wound repair, and disease pathogenesis, we disrupted the fibrinogen A alpha chain gene in mice. Homozygous, A alpha chain-deficient (A alpha-/-) mice are born normal in appearance, and there is no evidence of fetal loss of these animals based on the Mendelian pattern of transmission of the mutant A alpha chain allele. All of the component chains of fibrinogen (A alpha, B beta, and gamma) are immunologically undetectable in the circulation of both neonatal and adult A alpha-/- mice, and blood samples fail to either clot or support platelet aggregation in vitro. Overt bleeding events develop shortly after birth in approximately 30% of A alpha-/- mice, most frequently in the peritoneal cavity, skin, and soft tissues around joints. Remarkably, most newborns displaying signs of bleeding ultimately control the loss of blood, clear the affected tissues, and survive the neonatal period. Juveniles and young adult A alpha-/- mice are predisposed to spontaneous fatal abdominal hemorrhage, but long-term survival is variable and highly dependent on genetic background. The periodic rupture of ovarian follicles in breeding-age A alpha-/- females does not appear to significantly diminish life expectancy relative to males; however, pregnancy uniformly results in fatal uterine bleeding around the tenth day of gestation. Microscopic analysis of spontaneous lesions found in A alpha-/- mice suggests that fibrin(ogen) plays a fundamental role in the organization of cells at sites of injury.

Afibrinogenemia↗

PubMed2003

Annotating large genomes with exact word matches.

We have developed a tool for rapidly determining the number of exact matches of any word within large, internally repetitive genomes or sets of genomes. Thus we can readily annotate any sequence, including the entire human genome, with the counts of its constituent words. We create a Burrows-Wheeler transform of the genome, which together with auxiliary data structures facilitating counting, can reside in about one gigabyte of RAM. Our original interest was motivated by oligonucleotide probe design, and we describe a general protocol for defining unique hybridization probes. But our method also has applications for the analysis of genome structure and assembly. We demonstrate the identification of chromosome-specific repeats, and outline a general procedure for finding undiscovered repeats. We also illustrate the changing contents of the human genome assemblies by comparing the annotations built from different genome freezes.

Algorithms↗

PubMed2000

Comparison of global transcription responses allows identification of Vibrio cholerae genes differentially expressed following infection.

Comparison of global transcription profiles of Vibrio cholerae grown in vitro and in vivo revealed that 20% of the genome was repressed and about 5% was induced under in vivo conditions. Hybridization with the cloned genes revealed that the virulence genes ctx, toxR, toxT and tcpA were induced under in vivo conditions. Dissection of two in vivo induced cosmids identified another set of three genes homologous to che Y1 involved in motility and chemotaxis, pnuC encoding the major component of the nicotinamide mononucleotide transport system and icmF belonging to a cassette involved in multiplication inside host cells. These results demonstrate that the global transcription profile approach might be a powerful method for identification of differentially expressed transcripts under in vivo conditions.

Animals↗

PubMed2006

South-seeking magnetotactic bacteria in the Northern Hemisphere.

Magnetotactic bacteria contain membrane-bound intracellular iron crystals (magnetosomes) and respond to magnetic fields. Polar magnetotactic bacteria in vertical chemical gradients are thought to respond to high oxygen levels by swimming downward into areas with low or no oxygen (toward geomagnetic north in the Northern Hemisphere and geomagnetic south in the Southern Hemisphere). We identified populations of polar magnetotactic bacteria in the Northern Hemisphere that respond to high oxygen levels by swimming toward geomagnetic south, the opposite of all previously reported magnetotactic behavior. The percentage of magnetotactic bacteria with south polarity in the environment is positively correlated with higher redox potential. The coexistence of magnetotactic bacteria with opposing polarities in the same redox environment conflicts with current models of the adaptive value of magnetotaxis.

Colony Count, Microbial↗

PubMed2004

Effect of parenteral fluoroquinolone administration on persistence of vancomycin-resistant Enterococcus faecium in the mouse gastrointestinal tract.

We examined the effect of subcutaneous fluoroquinolone antibiotic administration on persistence and density of vancomycin-resistant Enterococcus faecium stool colonization in mice. Levofloxacin and ciprofloxacin did not promote colonization in comparison to saline controls, whereas moxifloxacin and gatifloxacin promoted persistent overgrowth in a dose-dependent fashion.

Amino Acid Sequence↗

PubMed1993

Evaluation of colilert-marine water for detection of total coliforms and Escherichia coli in the marine environment.

A test that allows for early detection of fecally contaminated coastal water would enhance public health protection. Colilert-Marine Water (Colilert-MW; Environetics, Branford, Conn.) is a rapid 24-h test that has recently been developed to detect total coliforms and Escherichia coli in coastal water. We performed a premarketing evaluation of the Colilert-MW product, testing it in parallel with the multiple tube fermentation (MTF) method for 86 coastal water samples in southern California. Statistical analysis was performed by using paired t tests and linear regression. Bacterial isolates were evaluated by biochemical and genetic analysis. The results of this study showed a strong correlation between the traditional MTF and the Colilert-MW method for detection of total coliforms (r = 0.95) and E. coli (r = 0.89) in ocean water samples. Paired t-test results indicated that the Colilert-MW and MTF were equivalent in detecting E. coli and that the Colilert-MW may be more sensitive in the detection of total coliforms. We conclude that Colilert-MW would be a useful tool with which to monitor coastal beach water.

Base Sequence↗

PubMed1993

Fate of Enterobacter cloacae JP120 and Alcaligenes eutrophus AEO106(pRO101) in soil during water stress: effects on culturability and viability.

A sandy loam soil near field capacity moisture content (psi = -0.050 MPa) or air dried (psi = -300 MPa) was inoculated with about 3 x 10(7) CFU of Enterobacter cloacae JP120 and Alcaligenes eutrophus AEO106(pRO101) per g and incubated in 40-g portions at 17 degrees C in closed or open Erlenmeyer flasks. In the field-moist soil, selective plating, direct viable counts, and DNA hybridization showed only minor changes in the numbers of E. cloacae and A. eutrophus cells with time (14 days), and the results obtained with the three detection methods generally agreed. In the air-dried soil, the majority of both bacteria were found as intact DNA-carrying cells that were neither culturable nor viable by the methods employed in this study. The numbers of culturable E. cloacae and A. eutrophus cells dropped to 10(5) and 10(2) CFU/g, respectively, 2 h after inoculation. Direct viable counts showed that only about 1% of the cells detected by immunofluorescence microscopy were viable, but a fraction of viable nonculturable cells of both bacteria was present. A. eutrophus did not tolerate desiccation as well as E. cloacae. Only a minor fraction of the two test organisms regained their culturability or viability after rewetting of the air-dried soil; the number of total heterotrophic culturable bacteria, however, increased more than 10-fold and reached 73% of the level found in the field-moist soil at day 14.

Alcaligenes↗

PubMed1994

Use of solid-phase PCR for enhanced detection of airborne microorganisms.

The solid-phase PCR (SP-PCR) was compared with a culture-based technique for the detection of aerosolized Escherichia coli DH1. Results with SP-PCR showed an increase in detection sensitivity over that of culture methods. Therefore, SP-PCR may be useful for the detection of airborne microorganisms which may be nonculturable because of aerosolization or sampling stress.

Air Microbiology↗

PubMed2000

Culture-dependent and culture-independent characterization of microbial assemblages associated with high-temperature petroleum reservoirs.

Recent investigations of oil reservoirs in a variety of locales have indicated that these habitats may harbor active thermophilic prokaryotic assemblages. In this study, we used both molecular and culture-based methods to characterize prokaryotic consortia associated with high-temperature, sulfur-rich oil reservoirs in California. Enrichment cultures designed for anaerobic thermophiles, both autotrophic and heterotrophic, were successful at temperatures ranging from 60 to 90 degrees C. Heterotrophic enrichments from all sites yielded sheathed rods (Thermotogales), pleomorphic rods resembling Thermoanaerobacter, and Thermococcus-like isolates. The predominant autotrophic microorganisms recovered from inorganic enrichments using H(2), acetate, and CO(2) as energy and carbon sources were methanogens, including isolates closely related to Methanobacterium, Methanococcus, and Methanoculleus species. Two 16S rRNA gene (rDNA) libraries were generated from total community DNA collected from production wellheads, using either archaeal or universal oligonucleotide primer sets. Sequence analysis of the universal library indicated that a large percentage of clones were highly similar to known bacterial and archaeal isolates recovered from similar habitats. Represented genera in rDNA clone libraries included Thermoanaerobacter, Thermococcus, Desulfothiovibrio, Aminobacterium, Acidaminococcus, Pseudomonas, Halomonas, Acinetobacter, Sphingomonas, Methylobacterium, and Desulfomicrobium. The archaeal library was dominated by methanogen-like rDNAs, with a lower percentage of clones belonging to the Thermococcales. Our results strongly support the hypothesis that sulfur-utilizing and methane-producing thermophilic microorganisms have a widespread distribution in oil reservoirs and the potential to actively participate in the biogeochemical transformation of carbon, hydrogen, and sulfur in situ.

Archaea↗

PubMed2001

Sphingomonas alaskensis strain AFO1, an abundant oligotrophic ultramicrobacterium from the North Pacific.

Numerous studies have established the importance of picoplankton (microorganisms of < or =2 microm in length) in energy flow and nutrient cycling in marine oligotrophic environments, and significant effort has been directed at identifying and isolating heterotrophic picoplankton from the world's oceans. Using a method of diluting natural seawater to extinction followed by monthly subculturing for 12 months, a bacterium was isolated that was able to form colonies on solid medium. The strain was isolated from a 10(5) dilution of seawater where the standing bacterial count was 3.1 x 10(5) cells ml(-1). This indicated that the isolate was representative of the most abundant bacteria at the sampling site, 1.5 km from Cape Muroto, Japan. The bacterium was characterized and found to be ultramicrosized (less than 0.1 microm(3)), and the size varied to only a small degree when the cells were starved or grown in rich media. A detailed molecular (16S rRNA sequence, DNA-DNA hybridization, G+C mol%, genome size), chemotaxonomic (lipid analysis, morphology), and physiological (resistance to hydrogen peroxide, heat, and ethanol) characterization of the bacterium revealed that it was a strain of Sphingomonas alaskensis. The type strain, RB2256, was previously isolated from Resurrection Bay, Alaska, and similar isolates have been obtained from the North Sea. The isolation of this species over an extended period, its high abundance at the time of sampling, and its geographical distribution indicate that it has the capacity to proliferate in ocean waters and is therefore likely to be an important contributor in terms of biomass and nutrient cycling in marine environments.

Base Composition↗