The species-specific mode of action of the antimicrobial peptide subtilosin against Listeria monocytogenes Scott A.

AIMS: To elucidate the molecular mechanism of action of the antimicrobial peptide subtilosin against the foodborne pathogen Listeria monocytogenes Scott A. METHODS AND RESULTS: Subtilosin was purified from a culture of Bacillus amyloliquefaciens. The minimal inhibitory concentration of subtilosin against L. monocytogenes Scott A was determined by broth microdilution method. The effect of subtilosin on the transmembrane electrical potential (ΔΨ) and pH gradient (ΔpH), and its ability to induce efflux of intracellular ATP, was investigated. Subtilosin fully inhibited L. monocytogenes growth at a concentration of 19 µg ml(-1) . Subtilosin caused a partial depletion of the ΔΨ and had a similar minor effect on the ΔpH. There was no significant efflux of intracellular ATP. CONCLUSION: Subtilosin likely acts upon L. monocytogenes Scott A by perturbing the lipid bilayer of the cellular membrane and causing intracellular damage, leading to eventual cell death. Subtilosin's mode of action against L. monocytogenes Scott A differs from the one previously described for another human pathogen, Gardnerella vaginalis. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report on the specific mode of action of subtilosin against L. monocytogenes and the first report of a bacteriocin with a species-specific mode of action.

The aetiology of bacterial vaginosis.

Bacterial vaginosis (BV) is the most common vaginal infection among women of childbearing age. This condition is notorious for causing severe complications related to the reproductive health of women. Five decades of intense research established many risk factors for acquisition of BV; however, because of the complexity of BV and lack of a reliable animal model for this condition, its exact aetiology remains elusive. In this manuscript, we use a historical perspective to critically review the development of major theories on the aetiology of BV, ultimately implicating BV-related pathogens, healthy vaginal microbiota, bacteriophages and the immune response of the host. None of these theories on their own can reliably explain the epidemiological data. Instead, BV is caused by a complex interaction of multiple factors, which include the numerous components of the vaginal microbial ecosystem and their human host. Many of these factors are yet to be characterized because a clear understanding of their relative contribution to the aetiology of BV is pivotal to the formulation of an effective treatment for and prophylaxis of this condition.

Postsurgical orthotic devices.

Custom-made orthotic devices are beneficial as preoperative and postoperative modalities. The decision to institute conservative treatment such as casting and orthotic appliances depends largely on the pathologic condition the patient presents with, the overall medical and social status of the patient, and the desired results the physician wants to achieve. Orthoses, prostheses, and bracing have evolved from tedious and labor-intensive leather and metal components to technically advanced plastics, composites, and computer-generated efficient and patient-friendly modalities. With the incorporation of human biomechanics and engineering as well as the introduction of human performance laboratories and the shared knowledge from these disciplines, the field of orthoses and prostheses continues to expand for the benefit of those who must rely on man-made devices to augment daily living activities.

The use of orthotic devices in adult acquired flatfoot deformity.

PTT dysfunction is the most common cause of adult acquired flat foot deformity. The aggressive nonoperative approach has become accepted more widely, in part because of the advances in orthotic and bracing technology and options. Many patients with a PTT dysfunction can be treated effectively with conservative management protocols. The goal of alleviating pain and correcting deformities is being accomplished with the proper application of the wide spectrum of orthotic modalities available today.

Biomechanical evaluation of the ability of casts and braces to immobilize the ankle and hindfoot.

We evaluated the ability of seven devices to immobilize a prosthetic ankle-foot complex against plantarflexion, dorsiflexion, inversion, and eversion forces: two casts (plaster of Paris and Fiberglas) and five removable braces (molded ankle/foot orthosis, composite boot brace, pneumatic boot walker, nonarticulating fracture boot, and ankle stirrup). Each device was applied to a prosthetic ankle-foot complex and evaluated on a test frame for resistance to sagittal motion and coronal torque. Results showed that casts offered significantly (P < or = 0.05) more resistance to motion in all directions tested than did the braces. The resistance offered by the devices tested depends on the conformity of the device to the shape of the foot in that plane and the material properties of the device. Braces offer the advantage of being easily removed and reapplied. Different braces offer specific advantages and disadvantages in different planes tested, and immobilization selection should be individualized based on this information.

Liposome-mediated DNA uptake and transient expression in Thermotoga.

We report here the successful application of a PCR-based method to detect genetic transformation of Thermotoga neapolitana and Thermotoga maritima. Plasmid vectors were constructed using pRQ7, an 846-bp plasmid found in Thermotoga species strain RQ7, which replicates by a rolling circle mechanism. The vector pJY1 was constructed by placing a gene encoding a thermostable chloramphenicol acetyltransferase from Stacphylococcus aureus under the control of the tac promoter and joining this with pRQ7 in a pBluescript vector. A second vector, pJY2, was similarly constructed using a gene encoding a kanamycin nucleotidyltransferase previously engineered for thermostability. Genetic transformation of T. neapolitana and T. maritima spheroplasts was achieved using cationic liposomes. The transforming DNA was detected in cells grown in liquid cultures using polymerase chain reaction amplification of the cat or kan genes. T. neapolitana could maintain pJY1 for at least 25 generations in liquid medium containing chloramphenicol. The pJY2 vector conferred kanamycin resistance to T. maritima cells grown in liquid culture. Isolation of stable transformants on solid media after 2-3 days of incubation at 77 degrees C was not possible with either vector, probably because of the instability of both vectors and antibiotics under these conditions. However, this transformation procedure provides, for the first time, a method to introduce DNA into this hyperthermophilic bacterium for potential applications such as targeted gene disruption analyses.

Differential gene expression in Thermotoga neapolitana in response to growth substrate.

We have previously shown that beta-galactosidase activity expressed in Thermotoga neapolitana cells grown on lactose is subject to repression by glucose when they are grown on both substrates whereas beta-galactosidase and beta-glucosidase activities observed in cells grown on cellobiose are not repressed by growth on both glucose and cellobiose. To examine the differential expression of bgalA, bgalB, bglA and bglB in T. neapolitana, total RNA was isolated from cells growing on either glucose, lactose or cellobiose as the sole source of carbon and transcripts encoding these genes were quantitated by Northern blot analyses. BglA expression was induced by cellobiose while bglB was expressed under all three conditions at a lower level. Expression of the beta-galactosidase genes, bgalA and bgalB, was detected only in lactose-grown cells. beta-Glucosidase enzyme activity was only found in cell extracts of cellobiose-grown cells while beta-galactosidase activity was found in both lactose- and cellobiose-grown cell extracts. Our results show that in cellobiose-grown cells, the high beta-glucosidase activity is likely due to expression of bglA and that neither bgalA nor bgalB is responsible for the beta-galactosidase activity.

Membrane-associated redox activities in Thermotoga neapolitana.

Elemental sulfur reduction by the hyperthermophilic bacterium Thermotoga neapolitana provides an alternative to hydrogen evolution during fermentation. Electrons are transferred from reduced cofactors (ferredoxin and NADH) to sulfur by a series of unknown steps. One enzyme that may be involved is an NADH:methyl viologen oxidoreductase (NMOR), an activity that in other fermenting organisms is associated with NADH:ferredoxin oxidoreductase. We found that 83% of NMOR activity was contained in the pellet fraction of cell extracts subjected to ultracentrifugation. This pellet fraction, presumably containing cell membranes, was required for electron transfer to NAD+ from ferredoxin-dependent pyruvate oxidation. However, the NMOR activity in this fraction used neither Thermotoga nor clostridial ferredoxins as substrates. NMOR activity was also detected in aerobically prepared vesicles. By comparison with ATPase activities, NMOR was found primarily on the cytoplasmic face of these vesicles. During these studies, an extracytoplasmic hydrogenase activity was discovered. In contrast to the soluble hydrogenase, this hydrogenase activity was completely inhibited when intact cells were treated with cupric chloride and was present on the extracytoplasmic face of vescides. In contrast to a soluble hydrogenase reported in Thermotoga maritima, this activity was air-stable and was inhibited by low concentrations of nitrite.

Exposure of Allan Hills 84001 and other achondrites on the Antarctic ice.

The enrichment of F on Antarctic meteorites is the result of their exposure to the atmosphere, and its measurement allows a subdivision of the terrestrial age into a duration of exposure on the ice and the time a meteorite was enclosed by the ice. In many cases, the periods of surface exposure are only small fractions of the terrestrial ages of meteorites collected in Antarctica. The enrichment of F on the surfaces of Antarctic achondrites was investigated by means of nuclear reaction analysis (NRA): scanning proton beams with an energy of 2.7 and 3.4 MeV were used to induce the reactions 19F(p, alpha gamma)16O and 19F(p,p gamma)19F, respectively. Gamma signals proportional to the F content were measured. The following Antarctic achondrites were investigated: Martian meteorite ALH 84001; diogenite ALHA77256; the eucrites ALHA81011 and ALHA78132; and in addition, the H5 chondrite ALHA79025. For ALH 84001, our data indicate a period of exposure on the ice of <500 years. Thus, this specimen was enclosed in the ice >95% of its terrestrial age of 13 000 years.

Differential expression and biological activity of the heparin-binding growth-associated molecule (HB-GAM) in lung cancer cell lines.

The growth of human lung cancer cells is regulated positively and negatively by a variety of growth factors through autocrine as well as paracrine mechanisms. In the present report, we studied the differential role and expression of a neuropolypeptide growth factor in 26 lung cancer cell lines. Expression of the heparin-binding growth-associated molecule (HB-GAM) in 12 small cell lung cancer (SCLC) cell lines was compared to that in 14 non-small cell lung cancer (NSCLC) cell lines. HB-GAM mRNA was expressed in 9 of 12 SCLC and 3 of 14 NSCLC cell lines as determined by RT-PCR analyses. Normal human bronchial epithelial cells were used as negative controls. All cell lines which expressed HB-GAM mRNA produced HB-GAM protein as well. Western blot analysis showed that the tumor cells secreted HB-GAM into the media. HB-GAM, purified from lung cancer cell lines, exerted biological activity on fibroblasts, endothelial cells and SW13 cells as determined by thymidine incorporation and soft agar cloning assays. In addition, the biological activity of HB-GAM was blocked by a specific antibody in a dose-dependent way. Our findings suggest that HB-GAM may serve as a marker for SCLC cell lines and that it may function as a paracrine growth factor in human lung cancer. HB-GAM may be a further member of the network of growth factors involved in proliferation, angiogenesis and metastasis of lung tumors.

Plasmid pRQ7 from the hyperthermophilic bacterium Thermotoga species strain RQ7 replicates by the rolling-circle mechanism.

The hyperthermophilic bacterium Thermotoga species strain RQ7 harbors an 846-bp plasmid, pRQ7, with a single open reading frame. Previously published analyses of the DNA sequence of pRQ7 suggested that it may replicate by a rolling-circle (RC) replication mechanism, and this report provides experimental evidence supporting this hypothesis. Single-stranded pRQ7 DNA accumulates in strain RQ7, as evidenced by the facts that this DNA bound to nitrocellulose membranes under nondenaturing conditions, was sensitive to S1 nuclease digestion, and hybridized to only one of two homologous DNA probes specific for each strand of the plasmid. The DNA encoding the open reading frame was cloned and expressed in Escherichia coli and gave a protein with a molecular mass of 26 kDa, similar to that deduced by sequence analysis. This protein bound to a fragment of pRQ7 that contains a putative double-stranded replication region in a magnesium-dependent reaction and made this fragment sensitive to S1 nuclease activity. It did not cause this same S1 nuclease sensitivity in the remainder of pRQ7. This activity on pRQ7 DNA suggests that this protein plays a role in plasmid replication.

Both DNA gyrase and reverse gyrase are present in the hyperthermophilic bacterium Thermotoga maritima.

Like all hyperthermophiles yet tested, the bacterium Thermotoga maritima contains a reverse gyrase. Here we show that it contains also a DNA gyrase. The genes top2A and top2B encoding the two subunits of a DNA gyrase-like enzyme have been cloned and sequenced. The Top2A (type II DNA topoisomerase A protein) is more similar to GyrA (DNA gyrase A protein) than to ParC [topoisomerase IV (Topo IV) C protein]. The difference is especially striking at the C-terminal domain, which differentiates DNA gyrases from Topo IV. DNA gyrase activity was detected in T. maritima and purified to homogeneity using a novobiocin-Sepharose column. This hyperhermophilic DNA gyrase has an optimal activity around 82-86 degrees C. In contrast to plasmids from hyperthermophilic archaea, which are from relaxed to positively supercoiled, we found that the plasmid pRQ7 from Thermotoga sp. RQ7 is negatively supercoiled. pRQ7 became positively supercoiled after addition of novobiocin to cell cultures, indicating that its negative supercoiling is due to the DNA gyrase of the host strain. The findings concerning DNA gyrase and negative supercoiling in Thermotogales put into question the role of reverse gyrase in hyperthermophiles.

Recent advances in genetic analyses of hyperthermophilic archaea and bacteria.

Hyperthermophilic Archaea and Bacteria are an extraordinarily important class of organisms for which genetic tools remain to be developed. Unique technological obstacles to this goal are posed by the thermophilic and, in some cases, strictly anaerobic nature of these organisms. However, recent advances in the cultivation of hyperthermophiles, in the discovery of genetic elements for vector development, and in the construction of genetic markers point toward the achievement of this goal in the near future. Transformation protocols have already been reported for Sulfolobus and Pyrococcus, and plasmid-mediated conjugation was recently found in Sulfolobus. Plasmids are available for Sulfolobus, Pyrococcus, and the bacterial hyperthermophile Thermotoga, and these provide the bases for vector construction in these hosts. A Desulfurococcus mobile intron may provide a novel means to introduce genes into a variety of archaeal hosts. With full genome sequences of several hyperthermophiles available soon, genetic tools will allow full exploitation of this information to study these organisms in depth and to utilize their unique properties in biotechnological applications.

Detection of ozone on Saturn's satellites Rhea and Dione.

The satellites Rhea and Dione orbit within the magnetosphere of Saturn, where they are exposed to particle irradiation from trapped ions. A similar situation applies to the galilean moons Europa, Ganymede and Callisto, which reside within Jupiter's radiation belts. All of these satellites have surfaces rich in water ice. Laboratory studies of the interaction of charged-particle radiation with water ice predicted the tenuous oxygen atmospheres recently found on Europa and Ganymede. However, theoretical investigations did not anticipate the trapping of significantly larger quantities of O2 within the surface ice. The accumulation of detectable abundances of O3, produced by the action of ultraviolet or charged-particle radiation on O2, was also not predicted before being observed on Ganymede. Here we report the identification of O3 in spectra of the saturnian satellites Rhea and Dione. The presence of trapped O3 is thus no longer unique to Ganymede, suggesting that special circumstances may not be required for its production.

Nuclear localization of insulin-like growth factor binding protein 3 in a lung cancer cell line.

Considerable evidence exists that lung cancer cell lines produce large amounts of insulin-like growth factor-binding proteins (IGFBPs). In addition, these cells are subject to an autocrine or paracrine growth control by insulin-like growth factors (IGFs). We now demonstrate by immunocytochemistry with IGFBP-3 antibodies that nuclei of a lung cancer cell line distinctly immunostain for IGFBP-3. This finding led us to investigate in more detail the localization of this protein that, to date, had only been known to occur extracellularly. Ligand blotting revealed that purified nuclear extracts contain a 43,000-Da IGFBP which can bind [I125]IGF-I. By Western blot this protein was identified as IGFBP-3. Thus, our data are consistent with the results of a previous structural study predicting a nuclear localization for IGFBP-3. Moreover, our findings raise the possibility that nuclear IGFBP-3 is functional and involved in the pathogenesis of lung cancer.

Titan's 5 micrometers spectral window: carbon monoxide and the albedo of the surface.

We have measured the spectrum of Titan near 5 micrometers and have found it to be dominated by absorption from the carbon monoxide 1-0 vibration-rotation band. The position of the band edge allows us to constrain the abundance of CO in the atmosphere and/or the location of the reflecting layer in the atmosphere. In the most likely case, 5 micrometers radiation is reflected from the surface and the mole fraction of CO in the atmosphere is qCO=10(+10/-5) ppm, significantly lower than previous estimates for tropospheric CO. The albedo of the reflecting layer is approximately 0.07(+0.02/-0.01) in the 5 micrometers continuum outside the CO band. The 5 micrometers albedo is consistent with a surface of mixed ice and silicates similar to the icy Galilean satellites. Organic solids formed in simulated Titan conditions can also produce similar albedos at 5 micrometers.

Insulin-like growth factors stimulate the release of insulin-like growth factor-binding protein-3 (IGFBP-3) and degradation of IGFBP-4 in nonsmall cell lung cancer cell lines.

Insulin-like growth factors (IGFs) are potent mitogens for lung cancer cells. Their proliferative activity is influenced by their binding proteins (IGFBPs). We report here on the regulatory effects of IGF-I and IGF-II on the production and release of IGFBPs by nonsmall cell lung cancer cell lines (NSCLC). The nine NSCLC cell lines used in this study showed messenger ribonucleic acid (mRNA) expression of all six IGFBPs known, as determined by PCR, and protein secretion of IGFBP-1, -2, -3, -4, and -5, as analyzed by Western immunoblots. The addition of IGFs to a serum-free medium showed divergent effects on IGFBP-3 and IGFBP-4 levels in a conditioned medium (CM). IGF-I and IGF-II, but not insulin, led to a much higher concentration of IGFBP-3 in the CM of all tested NSCLC cell lines, whereas the level of immunologically detected membrane-associated IGFBP-3 was decreased. Furthermore, Northern analysis of mRNA isolated from A549 revealed that IGFBP-3 specific mRNA was not changed by IGF-I or IGF-II, suggesting that the IGF-induced effects on IGFBP-3 depend on the release of cell-associated IGFBP-3. In contrast, IGFBP-4 levels were diminished by increasing concentrations of IGFs in the CM of the NSCLCs A549, NCI-H157, and U1752, with no response to insulin or the IGF-I analog, whereas IGFBP-4-specific mRNA was not changed by IGF-I or IGF-II, as determined by Northern analysis. The same effects were seen in a cell-free system after incubation of the CM with IGFs. The decrease in IGFBP-4 concentrations was prevented by coincubation of the CM with the IGFs and either ethylenediamine tetraacetate or 1,10-phenanthrolene, but not with other protease inhibitors. We suggest that IGFs may either activate an IGFBP-4-specific metalloprotease present in NSCLC CM or that the binding of IGFs to IGFBP-4 may enhance the susceptibility of IGFBP-4 to proteolytic degradation. Based on these data, we present evidence that IGFs may regulate their own availability both by releasing IGFBP-3 from cell membrances and through proteolytic degradation of IGFBP-4.

Detection of ozone on Ganymede.

An absorption band at 260 nanometers on the trailing hemisphere of Ganymede, identified as the Hartley band of Ozone (O3), was measured with the Hubble Space Telescope. The column abundance of ozone, 4.5 x 10(16) per square centimeter, can be produced by ion impacts or by photochemical equilibrium with previously detected molecular oxygen (O2). An estimated number density ratio of [O3]/[O2] = 10(-4) to 10(-3) requires an atmospheric density orders of magnitude higher than upper limits from spacecraft occultation experiments. Apparently, this O2-O3 "atmosphere" is trapped in Ganymede's surface ice, an inference consistent with the shift and broadening of the band compared with the gas-phase O3 band.

The hyperthermophilic bacterium Thermotoga neapolitana possesses two isozymes of the 3-phosphoglycerate kinase/triosephosphate isomerase fusion protein.

The phosphoglycerate kinase (pgk), triosephosphate isomerase (tpi), and enolase (eno) genes from Thermotoga neapolitana have been cloned and expressed in Escherichia coli. In high copy number, the pgk gene complemented an E. coli pgk- strain. In T. neapolitana, the pgk and tpi genes appear to be fused and eno is near those genes. Like T. maritima, T. neapolitaná produces phosphoglycerate kinase as both an individual enzyme and a fusion protein with triosephosphate isomerase, and triosephosphate isomerase activity is not found without associated phosphoglycerate kinase activity. Unlike T. maritima, which forms only a 70-kDa fusion protein, T. neapolitana expresses both 73-kDa and 81-kDa isozymes of this fusion protein. These isozymes are present in both T. neapolitana cells and in E. coli cells expressing T. neapolitana genes.