VirB6 is required for stabilization of VirB5 and VirB3 and formation of VirB7 homodimers in Agrobacterium tumefaciens.

VirB6 from Agrobacterium tumefaciens is an essential component of the type IV secretion machinery for T pilus formation and genetic transformation of plants. Due to its predicted topology as a polytopic inner membrane protein, it was proposed to form the transport pore for cell-to-cell transfer of genetic material and proteinaceous virulence factors. Here, we show that the absence of VirB6 leads to reduced cellular levels of VirB5 and VirB3, which were proposed to assist T pilus formation as minor component(s) or assembly factor(s), respectively. Overexpression of virB6 in trans restored levels of cell-bound and T pilus-associated VirB5 to wild type but did not restore VirB3 levels. Thus, VirB6 has a stabilizing effect on VirB5 accumulation, thereby regulating T pilus assembly. In the absence of VirB6, cell-bound VirB7 monomers and VirB7-VirB9 heterodimers were reduced and VirB7 homodimer formation was abolished. This effect could not be restored by expression of VirB6 in trans. Expression of TraD, a component of the transfer machinery of the IncN plasmid pKM101, with significant sequence similarity to VirB6, restored neither protein levels nor bacterial virulence but partly permitted T pilus formation in a virB6 deletion strain. VirB6 may therefore regulate T pilus formation by direct interaction with VirB5, and wild-type levels of VirB3 and VirB7 homodimers are not required.

Stabilization of subcritical bypass transition in the leading-edge boundary layer by suction

This work investigates the subcritical spatial transition in the swept Hiemenz boundary layer by means of direct numerical simulations (DNS). A pair of steady co-rotating vortices located at the attachment line is enforced as a primary disturbance leading to streaks which are stable. A small secondary, time-dependent disturbance interacts with these streaks such that instability and breakdown to turbulence may occur. The instability only occurs for a certain band of secondary disturbance frequencies. Positive secondary instability growth rates could be observed for Reynolds numbers as low as , whereas the linear critical Reynolds number is. Uniform wall suction is shown to stabilise this transition mechanism, analogously to results from linear stability theory. The effects of suction on the formation of primary streaks and on the secondary growth rate are decoupled. For streaks of different suction whose amplitude is held constant by adjusting the Reynolds number, the suction is shown to increase the growth rate of the secondary instability. The stabilising influence of wall suction consists in decreasing the streak amplitude only. Depending on the Reynolds number and the suction strength, breakdown may either occur locally and may be convected along the far-field streamlines, or occur globally and cover broad regions in the downstream direction.

ARD1 stabilization of TSC2 suppresses tumorigenesis via the mTOR signaling pathway

Mammalian target of rapamycin (mTOR) plays an important role in regulating various cellular functions, and the tuberous sclerosis 1 (TSC1)/TSC2 complex serves as a major repressor of the mTOR pathway. Here we demonstrated that arrest-defective protein 1 (ARD1) physically interacts with, acetylates, and stabilizes TSC2, thereby reducing mTOR activity. The inhibition of mTOR by ARD1 suppresses cell proliferation and increases autophagy, which further impairs tumorigenicity. Correlation between the levels of ARD1 and TSC2 was found in multiple tumor types, suggesting the physiological importance of ARD1 in stabilizing TSC2. Moreover, evaluation of loss of heterozygosity (LOH) at Xq28 revealed allelic loss in 31% of tested breast cancer cell lines and tumor samples. Together, our findings suggest that ARD1 functions as a negative regulator of the mTOR pathway and that dysregulation of the ARD1/TSC2/mTOR axis may contribute to cancer development. To further explore the signaling pathway of ARD1, we provided evidence showing the phosphorylation of ARD1 by IKKβ, which mediated the destabilization of ARD1. Future work may be needed to study the biological effect of this post-translational modification. ^

Stabilization of hillside in the work terraces of Alcaidesa in San Roque(Cádiz)

In the locality of The Hills of Alcaidesa in San Roque (Cádiz) has been constructed three blocks of housing called "The Terraces Alcaidesa" in a hillside in part virgin and in part with a refilling terrain without compaction with a width of 50 m and with a difference of levels between the most extreme points of 10 m.

Dynamic stabilization of L2 periodic orbits using attitude-orbit coupling effects

Numerical explorations show how the known periodic solutions of the Hill problem are modified in the case of the attitude-orbit coupling that may occur for large satellite structures. We focus on the case in which the elongation is the dominant satellite’s characteristic and find that a rotating structure may remain with its largest dimension in a plane parallel to the plane of the primaries. In this case, the effect produced by the non-negligible physical length is dynamically equivalent to the perturbation produced by an oblate central body on a mass-point satellite. Based on this, it is demonstrated that the attitude-orbital coupling of a long enough body may change the dynamical characteristics of a periodic orbit about the collinear Lagrangian points.

Dynamic stabilization of L2 periodic orbits using attitude-orbit coupling effects

Numerical explorations show how the known periodic solutions of the Hill problem are modified in the case of the attitude-orbit coupling that may occur for large satellite structures. We focus on the case in which the elongation is the dominant satellite?s characteristic and find that a rotating structure may remain with its largest dimension in a plane parallel to the plane of the primaries. In this case, the effect produced by the non-negligible physical dimension is dynamically equivalent to the perturbation produced by an oblate central body on a masspoint satellite. Based on this, it is demonstrated that the attitude-orbital coupling of a long enough body may change the dynamical characteristics of a periodic orbit about the collinear Lagrangian points.

Stabilization of embankments on inclined surfaces by micropiles

Embankments constructed on hillsides can have serious problems of stability, generally created by the action of water com-bined with the inclination of the hillside. In order to increase the stability or correct problems of instability already present, there are various methods that can be used: surface and deep drainage, reinforcements with anchored beams, medium and large diameter piles, etc. Standing out among these systems (for its versatility) is the use of micropiles which ?sew? the embankment to a non-unstable area of the hillside. This paper presents research undertaken by means of a finite element code for studying the effect and stress of the micropiles, comparing the results with real measurements taken in the south of Spain.

A molecular level picture of the stabilization of A-DNA in mixed ethanol–water solutions

Advances in computer power, methodology, and empirical force fields now allow routine “stable” nanosecond-length molecular dynamics simulations of DNA in water. The accurate representation of environmental influences on structure remains a major, unresolved issue. In contrast to simulations of A-DNA in water (where an A-DNA to B-DNA transition is observed) and in pure ethanol (where disruption of the structure is observed), A-DNA in ≈85% ethanol solution remains in a canonical A-DNA geometry as expected. The stabilization of A-DNA by ethanol is likely due to disruption of the spine of hydration in the minor groove and the presence of ion-mediated interhelical bonds and extensive hydration across the major groove.

Stabilization of microtubule dynamics by estramustine by binding to a novel site in tubulin: A possible mechanistic basis for its antitumor action

The cellular targets for estramustine, an antitumor drug used in the treatment of hormone-refractory prostate cancer, are believed to be the spindle microtubules responsible for chromosome separation at mitosis. Estramustine only weakly inhibits polymerization of purified tubulin into microtubules by binding to tubulin (Kd, ≈30 μM) at a site distinct from the colchicine or the vinblastine binding sites. However, by video microscopy, we find that estramustine strongly stabilizes growing and shortening dynamics at plus ends of bovine brain microtubules devoid of microtubule-associated proteins at concentrations substantially below those required to inhibit polymerization of the microtubules. Estramustine strongly reduced the rate and extent both of shortening and growing, increased the percentage of time the microtubules spent in an attenuated state, neither growing nor shortening detectably, and reduced the overall dynamicity of the microtubules. Significantly, the combined suppressive effects of vinblastine and estramustine on the rate and extent of shortening and dynamicity were additive. Thus, like the antimitotic mechanisms of action of the antitumor drugs vinblastine and taxol, the antimitotic mechanism of action of estramustine may be due to kinetic stabilization of spindle microtubule dynamics. The results may explain the mechanistic basis for the benefit derived from combined use of estramustine with vinblastine or taxol, two other drugs that target microtubules, in the treatment of hormone-refractory prostate cancer.

Phosphorylation of Ser-20 mediates stabilization of human p53 in response to DNA damage

Stabilization of p53 in response to DNA damage is caused by its dissociation from Mdm2, a protein that targets p53 for degradation in the proteasome. Dissociation of p53 from Mdm2 could be caused by DNA damage-induced p53 posttranslational modifications. The ATM and ATR kinases, whose activation in response to ionizing radiation (IR) and UV light, respectively, is required for p53 stabilization, directly phosphorylate p53 on Ser-15. However, phosphorylation of Ser-15 is critical for the apoptotic activity of p53 and not for p53 stabilization. Thus, whether any p53 modifications, and which, underlie disruption of the p53–Mdm2 complex after DNA damage remains to be determined. We analyzed the IR- and UV light-induced stabilization of p53 proteins with substitutions of Ser known to be posttranslationally modified after DNA damage. Substitution of Ser-20 was sufficient to abrogate p53 stabilization in response to both IR and UV light. Furthermore, both IR and UV light induced phosphorylation of p53 on Ser-20, which involved the majority of nuclear p53 protein and weakened the interaction of p53 with Mdm2 in vitro. ATM and ATR cannot phosphorylate p53 on Ser-20. We therefore propose that ATM and ATR activate an, as yet unidentified, kinase that stabilizes p53 by phosphorylating it on Ser-20.

Implications of a Developmental-Stage-Dependent Thylakoid-Bound Protease in the Stabilization of the Light-Harvesting Pigment-Protein Complex Serving Photosystem II during Thylakoid Biogenesis in Red Kidney Bean1

Intact etioplasts of bean (Phaseolus vulgaris) plants exhibit proteolytic activity against the exogenously added apoprotein of the light-harvesting pigment-protein complex serving photosystem II (LHCII) that increases as etiolation is prolonged. The activity increases in the membrane fraction but not in the stroma, where it remains low and constant and is mainly directed against LHCII and protochlorophyllide oxidoreductase. The thylakoid proteolytic activity, which is low in etioplasts of 6-d-old etiolated plants, increases in plants pretreated with a pulse of light or exposed to intermittent-light (ImL) cycles, but decreases during prolonged exposure to continuous light, coincident with chlorophyll (Chl) accumulation. To distinguish between the control of Chl and/or development on proteolytic activity, we used plants exposed to ImL cycles of varying dark-phase durations. In ImL plants exposed to an equal number of ImL cycles with short or long dark intervals (i.e. equal Chl accumulation but different developmental stage) proteolytic activity increased with the duration of the dark phase. In plants exposed to ImL for equal durations to such light-dark cycles (i.e. different Chl accumulation but same developmental stage) the proteolytic activity was similar. These results suggest that the protease, which is free to act under limited Chl accumulation, is dependent on the developmental stage of the chloroplast, and give a clue as to why plants in ImL with short dark intervals contain LHCII, whereas those with long dark intervals possess only photosystem-unit cores and lack LHCII.

Conformational change and enhanced stabilization of the vitamin D receptor by the 1,25-dihydroxyvitamin D3 analog KH1060.

The 1,25-dihydroxyvitamin D3 [1,25-(OH)2vitamin D3] analog KH1060 exerts very potent effects on cell proliferation and cell differentiation via the vitamin D receptor (VDR). However, the activities of KH1060 are not associated with an increased affinity for the VDR. We now show that increased stabilization of the VDR-KH1060 complex could be an explanation for its high potencies. VDR half-life studies performed with cycloheximide-translational blocked rat osteoblast-like ROS 17/2.8 cells demonstrated that, in the absence of ligand, VDR levels rapidly decreased. After 2 hr, less than 10% of the initial VDR level could be measured. In the presence of 1,25-(OH)2vitamin D3, the VDR half-life was 15 hr. After 24 hr. less than 20% of the initial VDR content was detectable, whereas, at this time-point, when the cells were incubated with KH1060 80% of the VDR was still present. Differences in 1,25-(OH)2vitamin D3- and KH1060-induced conformational changes of the VDR could underlie the increased VDR stability. As assessed by limited proteolytic digestion analysis, both 1,25-(OH)2vitamin D3 and KH1060 caused a specific conformational change of the VDR. Compared with 1,25-(OH)2vitamin D3, KH1060 induced a conformational change that led to a far more dramatic protection of the VDR against proteolytic degradation. In conclusion, the altered VDR stability and the possibly underlying change in VDR conformation caused by KH1060 could be an explanation for its enhanced bioactivity.

Stabilization of diverged tandem repeats by mismatch repair: evidence for deletion formation via a misaligned replication intermediate.

A functional methyl-directed mismatch repair pathway in Escherichia coli prevents the formation of deletions between 101-bp tandem repeats with 4% sequence divergence. Deletions between perfectly homologous repeats are unaffected. Deletion in both cases occurs independently of the homologous recombination gene, recA. Because the methyl-directed mismatch repair pathway detects and excises one strand of a mispaired duplex, an intermediate for RecA-independent deletion of tandem repeats must therefore be a heteroduplex formed between strands of each repeat. We find that MutH endonuclease, which in vivo incises specifically the newly replicated strand of DNA, and the Dam methylase, the source of this strand-discrimination, are required absolutely for the exclusion of "homeologous" (imperfectly homologous) tandem deletion. This supports the idea that the heteroduplex intermediate for deletion occurs during or shortly after DNA replication in the context of hemi-methylation. Our findings confirm a "replication slippage" model for deletion formation whereby the displacement and misalignment of the nascent strand relative to the repeated sequence in the template strand accomplishes the deletion.

Stabilization of tetanus and diphtheria toxoids against moisture-induced aggregation.

The progress toward single-dose vaccines has been limited by the poor solid-state stability of vaccine antigens within controlled-release polymers, such as poly(lactide-co-glycolide). For example, herein we report that lyophilized tetanus toxoid aggregates during incubation at 37 degrees C and elevated humidity--i.e., conditions relevant to its release from such systems. The mechanism and extent of this aggregation are dependent on the moisture level in the solid protein, with maximum aggregation observed at intermediate moisture contents. The main aggregation pathway is consistent with formaldehyde-mediated cross-linking, where reactive electrophiles created and stored in the vaccine upon formalinization (exposure to formaldehyde during vaccine preparation) react with nucleophiles of a second vaccine molecule to form intermolecular cross-links. This process is inhibited by the following: (i) succinylating the vaccine to block reactive amino groups; (ii) treating the vaccine with sodium cyanoborohydride, which presumably reduces Schiff bases and some other electrophiles created upon formalinization; and (iii) addition of low-molecular-weight excipients, particularly sorbitol. The moisture-induced aggregation of another formalinized vaccine, diphtheria toxoid, is also retarded by succinylation, suggesting the generality of this mechanism for formalinized vaccines. Hence, mechanistic stability studies of the type described herein may be important for the development of effective single-dose vaccines.