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PubMed1994

Immunofluorescence localization of the unconventional myosin, Myo2p, and the putative kinesin-related protein, Smy1p, to the same regions of polarized growth in Saccharomyces cerevisiae.

Myo2 protein (Myo2p), an unconventional myosin in the budding yeast Saccharomyces cerevisiae, has been implicated in polarized growth and secretion by studies of the temperature-sensitive myo2-66 mutant. Overexpression of Smy1p, which by sequence is a kinesin-related protein, can partially compensate for defects in the myo2 mutant (Lillie, S. H. and S. S. Brown, 1992. Nature (Lond.). 356:358-361). We have now immunolocalized Smy1p and Myo2p. Both are concentrated in regions of active growth, as caps at incipient bud sites and on small buds, at the mother-bud neck just before cell separation, and in mating cells as caps on shmoo tips and at the fusion bridge of zygotes. Double labeling of cells with either Myo2p or Smy1p antibody plus phalloidin was used to compare the localization of Smy1p and Myo2p to actin, and by extrapolation, to each other. These studies confirmed that Myo2p and Smy1p colocalize, and are concentrated in the same general regions of the cell as actin spots. However, neither colocalizes with actin. We noted a correlation in the behavior of Myo2p, Smy1p, and actin, but not microtubules, under a number of circumstances. In cdc4 and cdc11 mutants, which produce multiple buds, Myo2p and Smy1p caps were found only in the subset of buds that had accumulations of actin. Mutations in actin or secretory genes perturb actin, Smy1p and Myo2p localization. The rearrangements of Myo2p and Smy1p correlate temporally with those of actin spots during the cell cycle, and upon temperature and osmotic shift. In contrast, microtubules are not grossly affected by these perturbations. Although wild-type Myo2p localization does not require Smy1p, Myo2p staining is brighter when SMY1 is overexpressed. The myo2 mutant, when shifted to restrictive temperature, shows a permanent loss in Myo2p localization and actin polarization, both of which can be restored by SMY1 overexpression. However, the lethality of MYO2 deletion is not overcome by SMY1 overexpression. We noted that the myo2 mutant can recover from osmotic shift (unlike actin mutants; Novick, P., and D. Botstein. 1985. Cell. 40:405-416). We have also determined that the myo2-66 allele encodes a Lys instead of a Glu at position 511, which lies at an actin-binding face in the motor domain.

Actins↗
PubMed2003

Determination of bilayer thickness and lipid surface area in unilamellar dimyristoylphosphatidylcholine vesicles from small-angle neutron scattering curves: a comparison of evaluation methods.

Small-angle neutron scattering (SANS) experiments were performed on unilamellar 1,2-dimyristoylphosphatidylcholine (DMPC) vesicles prepared in heavy water by extrusion through polycarbonate filters with 500 A pores. The data obtained at 30+/-0.1 degrees C were evaluated using a five-strip function model of the bilayer coherent neutron scattering length density, three different approximate form factors describing scattering from vesicles, and different methods of evaluation of the experimental data. It is shown that the results obtained from the SANS data in the range of scattering vector values 0.0316 A(-1)< q<0.0775 A(-1) are not sensitive to the vesicle form factor, nor to the evaluation method. Using the hollow sphere model of vesicles convoluted with the Gaussian distribution of their sizes, a constrained bilayer polar region thickness of 9 A and a DMPC headgroup volume of 325.5 A(3), it was possible to obtain from the experimental data the DMPC surface area as 58.9+/-0.8 A(2), the bilayer thickness as 44.5+/-0.3 A and the number of water molecules as 6.8+/-0.2 per DMPC located in the bilayer polar region.

Algorithms↗
PubMed2005

Geology and insolation-driven climatic history of Amazonian north polar materials on Mars.

Mariner 9 and Viking spacecraft images revealed that the polar regions of Mars, like those of Earth, record the planet's climate history. However, fundamental uncertainties regarding the materials, features, ages and processes constituting the geologic record remained. Recently acquired Mars Orbiter Laser Altimeter data and Mars Orbiter Camera high-resolution images from the Mars Global Surveyor spacecraft and moderately high-resolution Thermal Emission Imaging System visible images from the Mars Odyssey spacecraft permit more comprehensive geologic and climatic analyses. Here I map and show the history of geologic materials and features in the north polar region that span the Amazonian period (approximately 3.0 Gyr ago to present). Erosion and redeposition of putative circumpolar mud volcano deposits (formed by eruption of liquefied, fine-grained material) led to the formation of an Early Amazonian polar plateau consisting of dark layered materials. Crater ejecta superposed on pedestals indicate that a thin mantle was present during most of the Amazonian, suggesting generally higher obliquity and insolation conditions at the poles than at present. Brighter polar layered deposits rest unconformably on the dark layers and formed mainly during lower obliquity over the past 4-5 Myr (ref. 20). Finally, the uppermost layers post-date the latest downtrend in obliquity <20,000 years ago.

Journal Article↗
PubMed1995

Effect of uterine space and fetal sex on conceptus development and in vitro release of progesterone and estrone from regions of the porcine placenta throughout gestation.

Intact (n = 17) and unilaterally hysterectomized-ovariectomized (UHOX, n = 19) gilts were used to study conceptus development and the steroidogenic capability of three regions of the porcine placenta under normal and crowded intrauterine conditions. Gilts were hysterectomized on either Days 30, 50, 70, or 90 of gestation. Placentas were combined according to the sex of the fetus associated with the placental unit, except at Day 30, when all were combined. Placentas were cut into three regions: middle, inner, and polar. Placental tissues were incubated for 0, 1, 5, and 3 hr, and concentrations of progesterone (P4) and estrone (E1) in the incubation medium were determined. Fetal survival rate was greater (P < 0.01, 0.05, and 0.001 at days 50, 70, and 90, respectively) in intact than in UHOX gilts. Placental length and weight, fetal length and weight, and allantoic fluid volume were greater (P < 0.05, 0.001, 0.05, and 0.001, respectively) in intact than in UHOX gilts. P4 concentration was lower (P < 0.001, 0.002, and 0.001 at Days 50, 70, and 90, respectively) in the incubation medium of polar region placenta compared with the other regions. The release of P4 was greater (P < 0.005) from the placentas of intact versus UHOX gilts at Day 90 but was not different at Days 30, 50, or 70. The sex of the fetus did not affect placental P4 release. The region of the placenta affected E1 release at Day 30 (P < 0.01, greater in polar section) and Day 50 (P < 0.06, less in polar section). There was no effect of uterine status on E1 release.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMedDate not supplied

Response of the mouse lens to varying sizes of injured area.

PURPOSE: To examine the response of the lens to varying sizes of perforating injury. METHODS: Four-week-old mice were used. Injuries consisted of pricking in the central region of the lens by transcorneal insertion of needles of two different sizes. After injury, the eyeballs were removed sequentially at various intervals up to 30 days and examined morphologically. RESULTS: The mouse lens showed three patterns of reaction; retained transparency, posterior opacity, and anterior opacity. (1) When the lens remained clear, the injury was small in area. The damaged portion of the lens was repaired by epithelial proliferation. (2) When opacity occurred abruptly at the posterior cortical area, epithelial damage was mild and lens fiber damage was relatively severe. Evans blue dye moved toward the posterior polar region along the cortical fiber arrangement. (3) When opacity developed abruptly at the anterior cortex, the size of epithelial damage and the damage to lens fibers were extensive. The posterior cortex remained transparent. Evans blue dye remained in the anterior polar region just beneath the injured part. CONCLUSION: It was found that the size of the injured area is a determinant of repair or opacity, and the site of opacity is dependent on the severity of epithelial damage and the location of the liquefied area.

Animals↗
PubMed2006

Diapir-induced reorientation of Saturn's moon Enceladus.

Enceladus is a small icy satellite of Saturn. Its south polar region consists of young, tectonically deformed terrain and has an anomalously high heat flux. This heat flux is probably due to localized tidal dissipation within either the ice shell or the underlying silicate core. The surface deformation is plausibly due to upwelling of low-density material (diapirism) as a result of this tidal heating. Here we show that the current polar location of the hotspot can be explained by reorientation of the satellite's rotation axis because of the presence of a low-density diapir. If the diapir is in the ice shell, then the shell must be relatively thick and maintain significant rigidity (elastic thickness greater than approximately 0.5 km); if the diapir is in the silicate core, then Enceladus cannot possess a global subsurface ocean, because the core must be coupled to the overlying ice for reorientation to occur. The reorientation generates large (approximately 10 MPa) tectonic stress patterns that are compatible with the observed deformation of the south polar region. We predict that the distribution of impact craters on the surface will not show the usual leading hemisphere-trailing hemisphere asymmetry. A low-density diapir also yields a potentially observable negative gravity anomaly.

Journal Article↗
PubMed1997

Morphogenetic potential of the chick leg interdigital mesoderm when diverted from the cell death program.

There is evidence that the interdigital mesoderm may be in an undifferentiated state. For example, under experimental manipulation in vivo it may be diverted from cell death to digit formation. In the present work we wanted to analyze the maximum morphogenetic potential of the interdigital cells. To do this we made recombinant limbs of three types, the first using dissociated-reaggregated leg interdigital mesoderm, the second using the same tissue but without dissociation and the third adding a piece of polarizing region to the dissociated interdigit. In all three the massive cell death of the interdigit failed to occur. The first type of recombinant formed a small nodule of cartilage while the other two formed a well-developed digit. Our data indicate that the maximum morphogenetic potential of the interdigital tissue appears constrained to form digits and that dissociation of the tissue decreased this ability; polarizing region restores the ability of dissociated cell recombinants to form a digit. We also analyzed in these recombinants the expression of a battery of genes implicated in interdigital cell death or in digital morphogenesis. The pattern of expression of each gene analyzed was identical in the three types of recombinant limbs. The expression of Msx1 and Msx2 genes was maintained under the ridge indicating a good interaction between the interdigital cells, both dissociated and undissociated, and the apical ridge. The expression of Hoxd-12, Hoxd-13 and Hoxa-13 genes was maintained in the recombinants, indicating that these cells carry information about their autopodial origin, and this correlates well with their distal restricted morphogenetic potential. Finally, the patterns of expression of the Bmp-2, Bmp-4 and Bmp-7 genes indicated that they are independently regulated in the recombinants and that Bmp-4 and Bmp-7 have wider expression domains than the areas of cell death that were only detected under the regressing apical ridge during day 3 of the experiment.

Animals↗
PubMed2002

Geographical and temporal variation in levels of organochlorine contaminants in marine mammals.

The interpretation of the spatial and temporal patterns of variation in organochlorine concentrations in marine mammal populations is complex because of the lack of wide-scale, long-term surveys. Therefore the results from several surveys must be combined and this causes undesired heterogeneity due to differences in the sampling and analytical techniques used and in the biological characteristics of the individuals sampled. Moreover, information is not homogeneously distributed in either space or in time. Most research is concentrated in western Europe, northern America and certain areas of Asia, while it is extremely limited or non-existent in Africa and most regions of the southern hemisphere. Marine mammals from the temperate fringe of the northern hemisphere, particularly fish-eating species which inhabit the mid-latitudes of Europe and North America, show the greatest organochlorine loads; noteworthy are the extremely high levels found in the Mediterranean Sea and certain locations on the western coasts of the United States. Concentrations in the tropical and equatorial fringe of the northern hemisphere and throughout the southern hemisphere are low or extremely low. The polar regions of both hemispheres showed the lowest concentrations of DDTs and PCBs, although levels of HCHs, chlordanes and HCB were moderate to high in the cold waters of the North Pacific. During recent decades, concentrations have tended to decrease in the regions where pollution was initially high but they have increased in regions located far from the pollution source as a consequence of atmospheric transport and redistribution. It is expected that the Arctic and, to a lesser extent, the Antarctic, will become major sinks for organochlorines in the future; this process may already be significant for some compounds such as HCB and HCHs. Effort should be devoted to both assessment of organochlorine trends in the now highly polluted populations of the temperate fringe of the northern hemisphere and to the implementation of long-term monitoring of marine mammal populations inhabiting polar regions.

Animals↗
PubMed2001

STEM Analysis of Caenorhabditis elegans muscle thick filaments: evidence for microdifferentiated substructures.

In the thick filaments of body muscle in Caenorhabditis elegans, myosin A and myosin B isoforms and a subpopulation of paramyosin, a homologue of myosin heavy chain rods, are organized about a tubular core. As determined by scanning transmission electron microscopy, the thick filaments show a continuous decrease in mass-per-length (MPL) from their central zones to their polar regions. This is consistent with previously reported morphological studies and suggests that both their content and structural organization are microdifferentiated as a function of position. The cores are composed of a second distinct subpopulation of paramyosin in association with the alpha, beta, and gamma-filagenins. MPL measurements suggest that cores are formed from seven subfilaments containing four strands of paramyosin molecules, rather than the two originally proposed. The periodic locations of the filagenins within different regions and the presence of a central zone where myosin A is located, implies that the cores are also microdifferentiated with respect to molecular content and structure. This differentiation may result from a novel "induced strain" assembly mechanism based upon the interaction of the filagenins, paramyosin and myosin A. The cores may then serve as "differentiated templates" for the assembly of myosin B and paramyosin in the tapering, microdifferentiated polar regions of the thick filaments.

Animals↗
PubMed1989

A reinterpretation of Na channel gating and permeation in terms of a phase transition between a transmembrane S4 alpha-helix and a channel-helix. A theoretical study.

A functional model for the S4/IV alpha-helix of the action potential sodium channel is described by means of a thermodynamic approach. The model is based on a phase transition between the alpha-helix and an ion conducting channel-helix which is similar to the well established helix-coil transition in solution. The right hand channel-helix is a peptide chain with an alternating sequence of torsional angles (phi1, psi1) = (87 degrees, 315 degrees) and (phi2, psi2) = (22 degrees, 107 degrees) which yields a helix of 13.5 A per turn. The axial dipole moments of the peptide bonds of this chain of L-amino acids nearly cancel each other out in a similar way to those in the gramicidin A channel, which is formed by alternating D- and L-amino acids. The helix, which does not contain any H-bonds, is stabilized by a helical file of water molecules which includes the permeating ion(s). This file turns around the channel-helix to form a relatively stable "double helix" structure which corresponds to the open channel. Since every third side chain in the S4/IV helix carries a positive charge their environments must be polarized. These polarized regions form a left hand screening-helix around the alpha-helix with 27 A per turn. If all H-bonds of the alpha-helix are broken and the internal alpha-carbon atom is considered as fixed, the outer ten residues leave the membrane while the internal ten residues form the channel-helix. In this configuration every positively charged side chain matches nearly exactly every second polarized region of the screening-helix leaving the three regions in-between exposed to the water file containing the ion(s). This further stabilizes the channel and agrees nicely with the idea of cationic selectivity. An analysis of the energetics of the alpha-helix-channel-helix transition showed that the voltage-independent part of the free energy per helix residue could well be close to 0 kcal/mol and thus be in the range where a transition could occur. Two voltage-dependent contributions were included: the break down of the considerable dipole of the alpha-helix and the outward shift of the positive charges of the side chains upon channel-helix formation. Taking into account the fact that the formation of an alpha-helix is a highly cooperative process the degree of voltage dependence of the probability of formation of a channel-helix proved to be in the same range as experimental values for the open probability of modified Na channels whose inactivation had been removed. With regard to gating currents, the model predicts that 2.74 positive charges are moved in an outward direction. Consequences of the model for other experimental findings are discussed.

Amino Acids↗
PubMed1985

A quantitative analysis of the effect of all-trans-retinoic acid on the pattern of chick wing development.

Small, positively charged beads that slowly release known amounts of all-trans-retinoic acid have been implanted below the apical ectodermal ridge at the anterior margin (opposite somite 16) of wing buds of 3 1/2 day-old chick embryos. The continuous release of retinoic acid is shown to create an anteroposterior concentration gradient of retinoic acid in the limb field that is stable with time, despite the fact that this compound is metabolized by the limb tissue. With beads that release increasing amounts of retinoic acid, the normal 234 digit pattern is progressively altered to a 2234, to a 32234, and then to a 432234 pattern. The tissue concentrations of all-trans-retinoic acid required to change the digit pattern in this way range between 1 and 25 nM. When the same amounts of retinoic acid are released from posteriorly implanted beads (placed below the apical ectodermal ridge opposite somite border 19/20 or somite 20), the normal digit pattern is unaffected. Implantations of beads that release all-trans-retinoic acid are thus identical in their effect to grafts of cells from the limb polarizing region, which cause similar dose-dependent changes in the digit pattern when grafted to the anterior margin of the bud (but not when grafted opposite somites 19 or 20). Because of the low concentrations of retinoic acid required for its biological effect, the graded response observed, and the fact that a concentration gradient is established across the limb field, all-trans-retinoic acid closely mimics the putative morphogen that has been postulated to be emitted by polarizing region cells during normal development.

Animals↗
PubMed1996

Evidence that Shh cooperates with a retinoic acid inducible co-factor to establish ZPA-like activity.

Patterning across the anteroposterior axis of the vertebrate limb bud involves a signal from the polarizing region, a small group of cells at the posterior margin of the bud. Retinoic acid (RA; Tickle, C., Alberts, B., Wolpert, L. and Lee, J. (1982) Nature 296, 554-566) and Sonic hedgehog (Shh; Riddle, R. D. Johnson, R. L., Laufer, E. and Tabin, C. J. (1993) Cell 25, 1401-1416; Chang, D. T., Lopez, A., von Kessler, D. P., Chiang, C., Simandl, B. K., Zhao, R., Seldin, M. F., Fallon, J. F. and Beachy, P. A. (1994 Development 120, 3339-3353) have been independently postulated as such signals because they can mimic the mirror image digit duplication obtained after grafting polarizing cells to the anterior of limb buds. Here we show that a embryonal carcinoma cell line, P19, transfected with a Shh expression vector shows low polarizing activity, but when cultured with retinoic acid, duplications like those induced by the polarizing region (ZPA) arise. Complete duplications are also obtained by cotransfecting P19 Shh cells with a constitutively active human retinoic acid receptor (VP16-hRARalpha). These data suggest that Shh and RA cooperate in generating ZPA activity and that Shh, while essential, may not act alone in this process.

Animals↗
PubMed2002

Measurement of atmospheric NO2 concentrations in Antarctica with NO2 filter badge and tube.

Nitrogen dioxide (NO2) levels in Antarctica were monitored to determine background levels and distribution profile. The NO2 levels were higher than those reported from tropical remote areas. The NO2 concentrations gradually increased proceeding from the edge of the continent to the polar region, suggesting global pollutant transportation. To avoid artificial contamination of the data, a sled was used because motor vehicles discharge high levels of NO2. NO2 tubes and filter badges were selected as monitoring devices because of their convenience of transportation in Antarctica. They are small in size, light and easy-to-handle, and usually employed in personal NO2 exposure measurements. Because of severe Antarctica climate, the devices needed to be protected from the bitter cold. For that purpose, we designed an insulation container to shield the devices. The fixed NO2 monitoring site was Patriot Hill, located on the coast of the Weddell Sea. We also measured NO2 concentration along the expedition route from the fixed site to the South Pole, along with the 80 degrees west longitude. The survey periods were during November 1992 and January 1993. NO2 concentrations in Antarctica ranged from 1.0 to 2.8 ppb, which were relatively higher than previously reported background concentrations. The NO2 concentrations increased when nearing to the South Pole, supporting the theory that atmospheric circulation converges in the polar regions. This indicated that air pollutants discharged in populated regions were widely spread to isolated areas, like the South Pole.

Air Pollutants↗
PubMed2004

Incidence of psychiatric disorders after extended residence in Antarctica.

OBJECTIVES: The incidence of psychiatric disorders and depressive symptoms was examined in a cohort of American men and women who spent an austral winter at two different research stations in Antarctica to determine whether extended residence of nonindigenous inhabitants in a polar region is associated with psychiatric morbidity. STUDY DESIGN: Debriefings interviews with 220 men and 93 women were conducted by 3 psychiatrists and 1 clinical psychologist at McMurdo Station and South Pole Station at the end of the austral winter between 1994 and 1997. Crewmembers were assigned a DSM-IV diagnosis if they satisfied diagnostic criteria. Debriefed crewmembers also completed the Structured Interview Guide for the Hamilton Depression Inventory-Seasonal Affective Disorders version (SIGH-SAD). RESULTS: Thirty-nine (12.5%) crewmembers presented with symptoms that met the criteria for one or more DSM-IV disorders. After weighting the prevalence to account for the low participation rate of civilian personnel, the incidence of DSM-IV disorders was 5.2%. Mood disorders were the most common diagnoses, accounting for 30.2% of all diagnoses, followed by adjustment disorders (27.9%), sleep-related disorders (20.9%), personality disorders (11.6%), and substance-related disorders (9.3%). Depressive symptoms as measured by the SAD-SIGH were significantly associated with female gender, military occupation, station, year of expedition, and DSM-IV diagnosis. CONCLUSION: Differences in the distribution of symptoms and diagnoses by demographic and expedition characteristics suggests that the social environment may be a more powerful determinant than the physical environment of psychiatric disorders in a polar region.

Adult↗
PubMed1985

Studies on the mechanism of retinoid-induced pattern duplications in the early chick limb bud: temporal and spatial aspects.

All-trans-retinoic acid causes striking digit pattern changes when it is continuously released from a bead implanted in the anterior margin of an early chick wing bud. In addition to the normal set of digits (234), extra digits form in a mirror-symmetrical arrangement, creating digit patterns such as a 432234. These retinoic acid-induced pattern duplications closely mimic those found after grafts of polarizing region cells to the same positions with regard to dose-response, timing, and positional effects. To elucidate the mechanism by which retinoic acid induces these pattern duplications, we have studied the temporal and spatial distribution of all-trans-retinoic acid and its potent analogue TTNPB in these limb buds. We find that the induction process is biphasic: there is an 8-h lag phase followed by a 6-h duplication phase, during which additional digits are irreversibly specified in the sequence digit 2, digit 3, digit 4. On average, formation of each digit seems to require between 1 and 2 h. The tissue concentrations, metabolic pattern, and spatial distribution of all-trans-retinoic acid and TTNPB in the limb rapidly reach a steady state, in which the continuous release of the retinoid is balanced by loss from metabolism and blood circulation. Pulse-chase experiments reveal that the half-time of clearance from the bud is 20 min for all-trans-retinoic acid and 80 min for TTNPB. Manipulations that change the experimentally induced steep concentration gradient of TTNPB suggest that a graded distribution of retinoid concentrations across the limb is required during the duplication phase to induce changes in the digit pattern. The extensive similarities between results obtained with retinoids and with polarizing region grafts raise the possibility that retinoic acid serves as a natural "morphogen" in the limb.

Animals↗
PubMed1995

Limb-patterning activity and restricted posterior localization of the amino-terminal product of Sonic hedgehog cleavage.

BACKGROUND: Sonic hedgehog (Shh), a vertebrate homolog of the Drosophila segment polarity gene hedgehog (hh), has been implicated in patterning of the developing chick limb. Such a role is suggested by the restricted expression of Shh along the posterior limb bud margin, and by the observation that heterologous cells expressing Shh have limb-polarizing activity resembling that of cells from the polarizing region of the posterior limb bud margin. It has not been demonstrated, however, that the Sonic hedgehog protein (SHH) alone is sufficient for limb patterning. SHH has been shown to undergo autoproteolytic cleavage in vitro, yielding two smaller products. It is of interest, therefore, to determine whether processing of SHH occurs in the developing limb and how such processing influences the function of SHH. RESULTS: We demonstrate that SHH is proteolytically processed in developing chick limbs. Grafts of cells expressing SHH protein variants that correspond to individual cleavage products demonstrate that the ability to induce patterned gene expression and to impose morphological pattern upon the limb bud is limited to the amino-terminal product (SHH-N) of SHH proteolytic cleavage. We also demonstrate that bacterially synthesized and purified SHH-N, released from implanted beads, is sufficient for limb-patterning activity. Finally, we show that the endogenous amino-terminal cleavage product is tightly localized to the posterior margin of the limb bud. CONCLUSIONS: Our data show that, of the two cleavage products resulting from SHH autoproteolysis, SHH-N expressed in grafted heterologous cells or supplied in purified form is sufficient to impose pattern upon the developing limb. Moreover, the restricted localization of the endogenous amino-terminal SHH cleavage product to the posterior border of the chick limb bud makes it unlikely that its patterning activity results from it being distributed in a broad gradient across the antero-posterior axis. More consistent with the observed localization is a model in which the amino-terminal SHH cleavage product exerts its patterning effects by local induction in or near the polarizing region, initiating a cascade of gene expression that ultimately extends across the developing limb.

Animals↗
PubMed2004

Investigation into the interaction of the bacterial protease OmpT with outer membrane lipids and biological activity of OmpT:lipopolysaccharide complexes.

Outer-membrane proteases T (OmpT) are important defence molecules of Gram-negative bacteria such as Escherichia coli found in particular in clinical isolates. We studied the interaction of OmpT with the membrane-forming lipids phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) from the inner leaflet and lipopolysaccharide (LPS) from the outer leaflet of the outer membrane. These investigations comprise functional aspects of the protein-lipid interaction mimicking the outer-membrane system as well as the bioactivity of LPS:OmpT complexes in the infected host after release from the bacterial surface. The molecular interaction of the lipids PE, PG, and LPS with OmpT was investigated by analysing molecular groups in the lipids originating from the apolar region (methylene groups), the interface region (ester), and the polar region (phosphates), and by analysing the acyl-chain melting-phase behaviour of the lipids. The activity of OmpT and LPS:OmpT complexes was investigated in biological test systems (human mononuclear cells and Limulus amoebocyte lysate assay) and with phospholipid model membranes. The results show a strong influence of OmpT on the mobility of the lipids leading to a considerable fluidization of the acyl chains of the phospholipids as well as LPS, and a rigidification of the phospholipid, but not LPS head groups. From this, a dominant role of the protein on the function of the outer membrane can be deduced. OmpT released from the outer membrane still contains slight contaminations of LPS, but its strong cytokine-inducing ability in mononuclear cells, which does not depend on the Toll-like receptors 2 and 4, indicates an LPS-independent mechanism of cell activation. This might be of general importance for infections induced by Gram-negative bacteria.

Animals↗