Self-reported menopausal symptoms in a racially diverse population and soy food consumption.

OBJECTIVES: Evaluate the association of self-reported vasomotor symptom (VMS) frequency with race/ethnicity among a diverse midlife US population and explore menopause symptom differences by dietary soy isoflavone (genistein+daidzein) consumption. STUDY DESIGN: Cross-sectional population-based study of peri- and postmenopausal women, ages 45-58. OUTCOMES: Recent VMS frequency, VMS ever; recent symptom bother (hot flashes, night sweats, headache and joint-ache). RESULTS: Of 18,500 potentially eligible women, 9325 returned questionnaires (50.4% response); 3691 were excluded (premenopausal, missing data, taking hormones). Of 5634 remaining women, 82.1% reported hot flashes ever, 73.1% reported night sweats ever; 48.8% and 38.6% reported recent hot flashes or night sweats, respectively. Compared with White women, Chinese, Japanese, Vietnamese, other Asian (each p<0.001) and Filipino (p<0.01) women less commonly reported ever having hot flashes; Asian women less commonly reported recent VMS bother (p<0.001). Black women more commonly reported hot flashes ever (p<0.05) and recent VMS bother (p<0.05). Compared with non-Hispanic White women, Hispanic women were less likely to report hot flashes (p<0.05) or night sweats (p<0.001) ever. Women were classified by isoflavone consumption: (1) none (n=1819), (2) 0.01-4.30 mg/day (n=1931), (3) 4.31-24.99 mg/day (n=1347) and (4) ≥ 25 mg/day (n=537). There were no group differences in recent VMS number/day: (1) 7.0 (95% CI 6.5, 7.5); (2) 6.4 (95% CI 6.0, 7.1); (3) 7.0 (95% CI 6.3, 8.2); and (4) 6.8 (95% CI 6.1, 7.7). CONCLUSIONS: Menopausal symptoms, independent of isoflavone intake, varied considerably by race/ethnicity and were least common among Asian races.

Formins and microtubules.

Formins have recently been recognized as prominent regulators of the microtubule (MT) cytoskeleton where they modulate the dynamics of selected MTs in interphase and mitosis. The association of formins with the MT cytoskeleton and their action on MT dynamics are relatively unexplored areas, yet growing evidence supports a direct role in their regulation of MT stability independent of their activity on actin. Formins regulate MT stability alone or in combination with accessory MT binding proteins that have previously been implicated in the stabilization of MTs downstream of polarity cues. As actin and MT arrays are typically remodeled downstream of signaling pathways that orchestrate cell shape and division, formins are emerging as excellent candidates for coordinating the responses of the cytoskeletal in diverse regulated and homeostatic processes.

Self-monitoring of behaviour as a risk reduction strategy for persons living with HIV.

To reduce the HIV-related transmission behaviours of persons living with HIV (PLH), a few efficacious interventions have been designed and evaluated. However, these interventions were delivered at relatively high cost, both in terms of time and resources. Given the challenges for health providers and community agencies in delivering these interventions, alternatives are needed. One possible intervention is allowing PLH to self-monitor their HIV transmission risk behaviour. Previous research suggests that self-monitoring of HIV-risk related behaviours may be a useful risk reduction strategy. This paper examines the impact of repeated risk assessments for behavioural self-monitoring as an intervention strategy for reducing sexual and substance use risk behaviours. A total of 365 PLH, recruited from community clinics, health management organizations, and health departments, completed self-assessments over time. Increased self-monitoring resulted in increases in protected sex with sexual partners of HIV-negative or unknown serostatus, and changes in attitudes conducive to reducing risk. Self-monitoring is a relatively low cost and easily implementable strategy for reducing the HIV-related transmission risk of PLH.

Interaction between EB1 and p150glued is required for anaphase astral microtubule elongation and stimulation of cytokinesis.

In animal cells, microtubules (MTs) of the mitotic apparatus (MA) communicate with the cell cortex to stimulate cytokinesis; however, the molecular nature of this stimulus remains elusive . A signal for cytokinesis likely involves the MT plus end binding family of proteins, which includes EB1, p150glued, APC, LIS1, and CLIP-170. These proteins modulate MT dynamics and facilitate interactions between growing MTs and their intracellular targets, including kinetochores, organelles, and the cell cortex . The dynein-dynactin complex mediates many of these microtubule capture events . We report that EB1 and p150glued interactions are required for stimulation of cytokinesis in dividing sea urchin eggs. Injected antibodies against EB1 or p150glued suppressed furrow ingression but did not prevent elongation of anaphase astral MTs toward the cortex, suggesting that EB1 and dynactin are both required for communication between the MA and the cortex. Targeted disruption of the interaction between EB1 and p150glued suppressed anaphase astral MT elongation and resulted in a delay of cytokinesis that could not be overcome by manipulation of the asters toward the cortex. We conclude that EB1 and dynactin participate in stimulation of the cleavage furrow, and their interaction promotes elongation of astral MTs at anaphase onset.

Synthesis of silicon-based infrared semiconductors in the Ge-Sn system using molecular chemistry methods.

Growth reactions based on a newly developed deuterium-stabilized Sn hydride [(Ph)SnD(3)] with Ge(2)H(6) produce a new family of Ge-Sn semiconductors with tunable band gaps and potential applications in high-speed, high-efficiency infrared optoelectronics. Metastable diamond-cubic films of Ge(1-x)Sn(x) alloys are created by chemical vapor deposition at 350 degrees C on Si(100). These exhibit unprecedented thermal stability and superior crystallinity despite the 17% lattice mismatch between the constituent materials. The composition, crystal structure, electronic structure, and optical properties of these materials are characterized by Rutherford backscattering, high-resolution electron microscopy, and X-ray diffraction, as well as Raman, IR, and spectroscopic ellipsometry. Electron diffraction reveals monocrystalline and perfectly epitaxial layers with lattice constants intermediate between those of Ge and alpha-Sn. X-ray diffraction in the theta-2theta mode shows well-defined peaks corresponding to random alloys, and in-plane rocking scans of the (004) reflection confirm a tightly aligned spread of the crystal mosaics. RBS ion-channeling including angular scans confirm that Sn occupies substitutional lattice sites and also provide evidence of local ordering of the elements with increasing Sn concentration. The Raman spectra show bands corresponding to Ge-Ge and Sn-Ge vibrations with frequencies consistent with random tetrahedral alloys. Resonance Raman and ellipsometry spectra indicate a band-gap reduction relative to Ge. The IR transmission spectra suggest that the band gap decreases monotonically with increasing Sn fraction. The synthesis, characterization, and gas-phase electron diffraction structure of (Ph)SnD(3) are also reported.

Cdc42, dynein, and dynactin regulate MTOC reorientation independent of Rho-regulated microtubule stabilization.

In migrating adherent cells such as fibroblasts and endothelial cells, the microtubule-organizing center (MTOC) reorients toward the leading edge [1-3]. MTOC reorientation repositions the Golgi toward the front of the cell [1] and contributes to directional migration [4]. The mechanism of MTOC reorientation and its relation to the formation of stabilized microtubules (MTs) in the leading edge, which occurs concomitantly with MTOC reorientation [3], is unknown. We show that serum and the serum lipid, lysophosphatidic acid (LPA), increased Cdc42 GTP levels and triggered MTOC reorientation in serum-starved wounded monolayers of 3T3 fibroblasts. Cdc42, but not Rho or Rac, was both sufficient and necessary for LPA-stimulated MTOC reorientation. MTOC reorientation was independent of Cdc42-induced changes in actin and was not blocked by cytochalasin D. Inhibition of dynein or dynactin blocked LPA- and Cdc42-stimulated MTOC reorientation. LPA also stimulates a Rho/mDia pathway that selectively stabilizes MTs in the leading edge [5, 6]; however, activators and inhibitors of MTOC reorientation and MT stabilization showed that each response was regulated independently. These results establish an LPA/Cdc42 signaling pathway that regulates MTOC reorientation in a dynein-dependent manner. MTOC reorientation and MT stabilization both act to polarize the MT array in migrating cells, yet these processes act independently and are regulated by separate Rho family GTPase-signaling pathways.

Spontaneous generation of stable pnictinyl radicals from "jack-in-the-box" dipnictines: a solid-state, gas-phase, and theoretical investigation of the origins of steric stabilization.

The molecular structures of the stable phosphinyl and arsinyl radicals, .PnR(2) [Pn = P (2); As (4); R = CH(SiMe(3))(2)], have been determined by gas-phase electron diffraction (GED) in conjunction with ab initio molecular orbital calculations. The X-ray crystal structures of the corresponding dipnictines, the "dimers", R(2)PnPnR(2) [Pn = P (1), As (3)], and the chloro derivatives R(2)PnCl [Pn = P (5), As (6)] have also been determined. Collectively, these structural investigations demonstrate that large distortions of the ligands attached to Pn occur when the pnictinyl radicals unite to form the corresponding dipnictine dimers. Principally, it is the shape and flexibility of the CH(SiMe(3))(2) ligands that permit the formation of the P-P and As-As bonds in 1and 3, respectively. However, theoretical studies indicate that in the process of pnictinyl radical dimerization to form 1 and 3, both molecules accumulate substantial amounts of potential energy and are thus primed to spring apart upon release from the solid state by melting, dissolution, or evaporation. The insights gleaned from these unusual systems have permitted a deeper understanding of the functioning of sterically demanding substituents.

mDia mediates Rho-regulated formation and orientation of stable microtubules.

Rho-GTPase stabilizes microtubules that are oriented towards the leading edge in serum-starved 3T3 fibroblasts through an unknown mechanism. We used a Rho-effector domain screen to identify mDia as a downstream Rho effector involved in microtubule stabilization. Constitutively active mDia or activation of endogenous mDia with the mDia-autoinhibitory domain stimulated the formation of stable microtubules that were capped and oriented towards the wound edge. mDia co-localized with stable microtubules when overexpressed and associated with microtubules in vitro. Rho kinase was not necessary for the formation of stable microtubules. Our results show that mDia is sufficient to generate and orient stable microtubules, and indicate that Dia-related formins are part of a conserved pathway that regulates the dynamics of microtubule ends.

Myopathy with tubulin-reactive crystalline inclusions.

A 61-year-old man with muscle aches and persistently elevated serum creatine kinase had aggregates of randomly oriented, rhomboidal or rectangular protein crystalline inclusions in the sarcoplasm of type II fibers. Immunochemical studies showed strong reactivity of the inclusions to tubulin antibodies, suggesting that these unique crystalline inclusions may be a consequence of altered synthesis, processing, or degradation of tubulin.

Detyrosinated (Glu) microtubules are stabilized by an ATP-sensitive plus-end cap.

Many cell types contain a subset of long-lived, 'stable' microtubules that differ from dynamic microtubules in that they are enriched in post-translationally detyrosinated tubulin (Glu-tubulin). Elevated Glu tubulin does not stabilize the microtubules and the mechanism for the stability of Glu microtubules is not known. We used detergent-extracted cell models to investigate the nature of Glu microtubule stability. In these cell models, Glu microtubules did not incorporate exogenously added tubulin subunits on their distal ends, while >70% of the bulk microtubules did. Ca(2+)-generated fragments of Glu microtubules incorporated tubulin, showing that Glu microtubule ends are capped. Consistent with this, Glu microtubules in cell models were resistant to dilution-induced breakdown. Known microtubule end-associated proteins (EB1, APC, p150(Glued) and vinculin focal adhesions) were not localized on Glu microtubule ends. ATP, but not nonhydrolyzable analogues, induced depolymerization of Glu microtubules in cell models. Timelapse and photobleaching studies showed that ATP triggered subunit loss from the plus end. ATP breakdown of Glu microtubules was inhibited by AMP-PNP and vanadate, but not by kinase or other inhibitors. Additional experiments showed that conventional kinesin or kif3 were not involved in Glu microtubule capping. We conclude that Glu microtubules are stabilized by a plus-end cap that includes an ATPase with properties similar to kinesins.

Efficiency of systematic sampling in histomorphometric bone research illustrated by hydroxyapatite-coated implants: optimizing the stereological vertical-section design.

The sampling efficiency of the unbiased stereological vertical-section method was analyzed in five hydroxyapatite-coated implants. They were inserted into humans and harvested after 1 year. To find an optimal sampling design for histomorphometric analyses, sampling efficiency was estimated by variance analyses at different sampling levels (humans, sections, fields of view, and number of counting items) and intensities. Only minor changes in variance were observed when the initial scheme was reduced to include just one of the two possible implant sides, every third field of view, and half the density of the probe; this reduced the total workload at the microscope to less than 10% for all sections. In addition, the number of sections for analysis could be reduced to every fourth section per implant (three to four sections for evaluation) without significantly increasing variance. The study demonstrated that biological variation contributed to the majority of the total observed variance. Optimizing the sampling design could significantly reduce the workload at the hard-tissue microtome and the microscope without reducing the quality of the data that were unbiased and that had low sampling variance as compared with the true biological variation.

Focal adhesion motility revealed in stationary fibroblasts.

Focal adhesions (FAs) are clustered integrins and associated proteins that mediate cell adhesion and signaling. A green fluorescent protein-beta1 integrin chimera was used to label FAs in living cells. In stationary cells, FAs were highly motile, moving linearly for several plaque lengths toward the cell center. FA motility was independent of cell density and resulted from contraction of associated actin fibers. In migrating cells, FAs were stationary and only moved in the tail. FA motility in stationary cells suggests that cell movement may be regulated by a clutch-like mechanism by which the affinity of integrins to substrate may be altered in response to migratory cues.

Detyrosination of tubulin regulates the interaction of intermediate filaments with microtubules in vivo via a kinesin-dependent mechanism.

Posttranslationally modified forms of tubulin accumulate in the subset of stabilized microtubules (MTs) in cells but are not themselves involved in generating MT stability. We showed previously that stabilized, detyrosinated (Glu) MTs function to localize vimentin intermediate filaments (IFs) in fibroblasts. To determine whether tubulin detyrosination or MT stability is the critical element in the preferential association of IFs with Glu MTs, we microinjected nonpolymerizable Glu tubulin into cells. If detyrosination is critical, then soluble Glu tubulin should be a competitive inhibitor of the IF-MT interaction. Before microinjection, Glu tubulin was rendered nonpolymerizable and nontyrosinatable by treatment with iodoacetamide (IAA). Microinjected IAA-Glu tubulin disrupted the interaction of IFs with MTs, as assayed by the collapse of IFs to a perinuclear location, and had no detectable effect on the array of Glu or tyrosinated MTs in cells. Conversely, neither IAA-tyrosinated tubulin nor untreated Glu tubulin, which assembled into MTs, caused collapse of IFs when microinjected. The epitope on Glu tubulin responsible for interfering with the Glu MT-IF interaction was mapped by microinjecting tubulin fragments of alpha-tubulin. The 14-kDa C-terminal fragment of Glu tubulin (alpha-C Glu) induced IF collapse, whereas the 36-kDa N-terminal fragment of alpha-tubulin did not alter the IF array. The epitope required more than the detyrosination site at the C terminus, because a short peptide (a 7-mer) mimicking the C terminus of Glu tubulin did not disrupt the IF distribution. We previously showed that kinesin may mediate the interaction of Glu MTs and IFs. In this study we found that kinesin binding to MTs in vitro was inhibited by the same reagents (i.e., IAA-Glu tubulin and alpha-C Glu) that disrupted the IF-Glu MT interaction in vivo. These results demonstrate for the first time that tubulin detyrosination functions as a signal for the recruitment of IFs to MTs via a mechanism that is likely to involve kinesin.

Microtubules and signal transduction.

Although molecular components of signal transduction pathways are rapidly being identified, how elements of these pathways are positioned spatially and how signals traverse the intracellular environment from the cell surface to the nucleus or to other cytoplasmic targets are not well understood. The discovery of signaling molecules that interact with microtubules (MTs), as well as the multiple effects on signaling pathways of drugs that destabilize or hyperstabilize MTs, indicate that MTs are likely to be critical to the spatial organization of signal transduction. MTs themselves are also affected by signaling pathways and this may contribute to the transmission of signals to downstream targets.

A phase II study of biweekly administration of paclitaxel in patients with recurrent epithelial ovarian cancer.

The purpose of this study was to assess the efficacy and toxicity of single agent paclitaxel administered biweekly to patients with relapse of epithelial ovarian cancer previously treated with platinum-based regimen. Forty patients received an initial paclitaxel dose of 134 mg/m2 administered intravenously over three hours every two weeks. 283 cycles were given. All 40 patients were evaluable for toxicity, which mainly consisted of granulocytopenia, myalgia/arthralgia, and peripheral neuropathy. Two patients developed severe hypersensitivity reactions. Dose escalation was possible by one level in 11 patients and by two levels in 12 patients, dose reductions were not necessary. Thirty-five patients were evaluated for response. Five obtained complete response (14%), eight obtained partial response (23%), and nine had stable disease (26%), while 11 patients showed progression (31%). The overall response rate was 37% (95% confidence interval 22-57%). The median duration of responses (complete and partial) was six months. Overall median time to progression and overall median survival for eligible patients (n = 35) was 4.3 months and 11 months, respectively. We conclude that biweekly administration of paclitaxel in recurrent epithelial ovarian carcinoma was active with manageable toxicity.

Relationship between microtubule dynamics and lamellipodium formation revealed by direct imaging of microtubules in cells treated with nocodazole or taxol.

Microtubules (MTs) contribute to the directional locomotion of many cell types through an unknown mechanism. Previously, we showed that low concentrations (<200 nM) of nocodazole or taxol reduced the rate of locomotion of NRK fibroblasts over 60% without altering MT polymer level [Liao et al., 1995: J. Cell Sci. 108:3473-3483]. In this paper, we directly measured the dynamics of MTs in migrating NRK cells injected with rhodamine tubulin and treated with low concentrations of nocodazole or taxol. Both drug treatments caused statistically significant reductions (approx. twofold) in growth and shortening rates and less dramatic effects on rescue and catastrophe transition frequencies. The percent time MTs were inactive (i.e., paused) increased greater than twofold in nocodazole- and taxol-treated cells, while the percent time growing was substantially reduced. Three parameters of MT dynamics were linearly related to the rates of locomotion determined previously: rate of shortening, percent time pausing and percent time growing. The number of MTs that came within 1 microm of the leading edge was reduced in drug-treated cells, suggesting that reduced MT dynamics may affect actin arrays necessary for cell locomotion. We examined two such structures, lamellipodium and adhesion plaques, and found that lamellipodia area was coordinately reduced with MT dynamics. No effect was detected on adhesion plaque density or distribution. In time-lapse recordings, MTs did not penetrate into the lamellipodium of untreated cells, suggesting that MTs affect lamellipodia either through their interaction with factors at the base of the lamellipodium or by releasing factors that diffuse into the lamellipodia. In support of the latter hypothesis, when all MTs were rapidly depolymerized by 20 microM nocodazole, we detected the rapid formation of exaggerated protrusions from the leading edge of the cell. Our results show for the first time a linear relationship between MT dynamics and the formation of the lamellipodium and support the idea that MT dynamics may contribute to cell locomotion by regulating the size of the lamellipodium, perhaps through diffusable factors.

Protein phosphatase 1 is targeted to microtubules by the microtubule-associated protein Tau.

Phosphorylation has been implicated in the regulation of microtubule (MT) stability and function by controlling the interactions between MTs and MT-associated proteins. We found previously that protein phosphatase inhibitors selectively break down stable MTs, suggesting that protein phosphatases may be involved in regulating MT stability. To identify the protein phosphatases involved, we examined purified calf brain MTs and found a protein phosphatase activity that copurified with MTs to constant stoichiometry. Western blot analysis and inhibitor profiles demonstrated that the MT-associated phosphatase was a type 1 protein phosphatase (PP1), which we named PP1MT. Recombinant PP1 catalytic subunit (PP1c) did not bind to MTs, whereas PP1MT did bind, suggesting the presence of proteins that target PP1 to MTs. By Sepharose CL-6B chromatography, the phosphatase activity of PP1MT eluted as a large protein complex of approximately 400 kDa. High salt (2 M NaCl) treatment followed by CL-6B chromatography dissociated PP1MT into PP1c and the MT-targeting subunit(s). The MT-targeting subunit was shown to be the MT-associated protein tau by PP1 blot overlays and other assays. Also, recombinant tau reconstituted the binding of PP1c to MTs. These results identify PP1 as the first tau binding protein and suggest that tau is a novel PP1-targeting subunit.