Loss of PTEN induces microtentacles through PI3K-independent activation of cofilin.

Loss of PTEN tumor suppressor enhances metastatic risk in breast cancer, although the underlying mechanisms are poorly defined. We report that homozygous deletion of PTEN in mammary epithelial cells induces tubulin-based microtentacles (McTNs) that facilitate cell reattachment and homotypic aggregation. Treatment with contractility-modulating drugs showed that McTNs in PTEN(-/-) cells are suppressible by controlling the actin cytoskeleton. Because outward microtubule extension is counteracted by actin cortical contraction, increased activity of actin-severing proteins could release constraints on McTN formation in PTEN(-/-) cells. One such actin-severing protein, cofilin, is activated in detached PTEN(-/-) cells that could weaken the actin cortex to promote McTNs. Expression of wild-type cofilin, an activated mutant (S3A), and an inactive mutant (S3E) demonstrated that altering cofilin phosphorylation directly affects McTNs formation. Chemical inhibition of PI3K did not reduce McTNs or inactivate cofilin in PTEN(-/-) cells. Additionally, knock-in expression of the two most common PI3K-activating mutations observed in human cancer patients did not increase McTNs or activate cofilin. PTEN loss and PI3K activation also caused differential activation of the cofilin regulators, LIM-kinase1 (LIMK) and Slingshot-1L (SSH). Furthermore, McTNs were suppressed and cofilin was inactivated by restoration of PTEN in the PTEN(-/-) cells, indicating that both the elevation of McTNs and the activation of cofilin are specific results arising from PTEN loss. These data identify a novel mechanism by which PTEN loss could remodel the cortical actin network to facilitate McTNs that promote tumor cell reattachment and aggregation. Using isogenic MCF-10A PTEN(-/-) and PIK3CA mutants, we have further demonstrated that there are clear differences in activation of cofilin, LIMK and SSH between PTEN loss and PI3K activation, providing a new evidence that these mutations yield distinct cytoskeletal phenotypes, which could have an impact on tumor biology.

c-Src differentially regulates the functions of microtentacles and invadopodia.

During metastasis, invading cells produce various actin-based membrane protrusions that promote directional migration and proteolysis of extracellular matrix (ECM). Observations of actin staining within thin, tubulin-based microtentacle (McTN) protrusions in suspended MDA-MB-231 tumor cells, prompted an investigation of whether McTNs are structural or functional analogs of invadopodia. We show here that MDA-MB-231 cells are capable of producing invadopodia and McTNs, both of which contain F-actin. Invadopodium formation was enhanced by the expression of a constitutively active c-Src kinase, and repressed by the expression of dominant-negative, catalytically inactive form of c-Src. In contrast, expression of inactive c-Src significantly increased McTN formation. Direct inhibition of c-Src with the SU6656 inhibitor compound also significantly enhanced McTN formation, but suppressed invadopodia, including the appearance of F-actin cores and phospho-cortactin foci, as well as completely blocking focal degradation of ECM. In addition, silencing of Tks5 in Src-transformed fibroblasts blocked invadopodia without affecting McTNs. Genetic modification of c-Src activity that promoted McTN formation augmented capillary retention of circulating tumor cells in vivo and rapid re-attachment of suspended cells in vitro, even though invadopodia were strongly suppressed. These results indicate that McTNs are capable of enhancing tumor cell reattachment, even in the absence of Tks5 and active Src, and define separate cytoskeletal mechanisms and functions for McTNs and invadopodia.

Metastatic breast tumors express increased tau, which promotes microtentacle formation and the reattachment of detached breast tumor cells.

The cytoskeletal organization of detached and circulating tumor cells (CTCs) is currently not well defined and may provide potential targets for new therapies to limit metastatic tumor spread. In vivo, CTCs reattach in distant tissues by a mechanism that is tubulin-dependent and suppressed by polymerized actin. The cytoskeletal mechanisms that promote reattachment of CTCs match exactly with the mechanisms supporting tubulin microtentacles (McTN), which we have recently identified in detached breast tumor cells. In this study, we aimed to investigate how McTN formation is affected by the microtubule-associated protein, tau, which is expressed in a subset of chemotherapy-resistant breast cancers. We demonstrate that endogenous tau protein localizes to McTNs and is both necessary and sufficient to promote McTN extension in detached breast tumor cells. Tau-induced McTNs increase reattachment of suspended cells and retention of CTCs in lung capillaries. Analysis of patient-matched primary and metastatic tumors reveals that 52% possess tau expression in metastases and 26% display significantly increased tau expression over disease progression. Tau enrichment in metastatic tumors and the ability of tau to promote tumor cell reattachment through McTN formation support a model in which tau-induced microtubule stabilization provides a selective advantage during tumor metastasis.

DNA fragmentation in leukocytes following repeated low dose sarin exposure in guinea pigs.

The objective of this study was to determine levels of DNA fragmentation in blood leukocytes and parietal cortex from guinea pigs following repeated low-level exposure to the chemical warfare nerve agent (CWNA) sarin. Guinea pigs were injected (s.c.) once a day for 10 days with saline, or 0.1, 0.2, or 0.4 LD50 (50% mean lethal dose) sarin dissolved in sterile physiological saline. Blood and parietal cortex was collected after injection at 0, 3, and 17 days recovery and evaluated for DNA fragmentation using single-cell gel electrophoresis (Comet assay). Cells were imaged using comet analysis software and three parameters of DNA fragmentation measured: tail length, percent DNA in the tail, and tail moment arm. Repeated low-dose exposure to sarin produced a dose-dependent response in leukocytes at 0 and 3 days post-exposure. There was a significant increase in all measures of DNA fragmentation at 0.2 and 0.4 LD50, but not at 0.1 LD50. There was no significant increase in DNA fragmentation in any of the groups at 17 days post-exposure. Sarin did not produce a systematic dose-dependent response in parietal cortex at any of the time points. However, significant increases in DNA fragmentation at 0.1 and 0.4 LD50 were observed at 0 and 3 days post-exposure. All measures of DNA fragmentation in both leukocytes and neurons returned to control levels by 17 days post-exposure, indicating a small and non-persistent increase in DNA fragmentation following repeated low-level exposure to sarin.

PAN-811 (3-aminopyridine-2-carboxaldehyde thiosemicarbazone), a novel neuroprotectant, elicits its function in primary neuronal cultures by up-regulating Bcl-2 expression.

Neurotoxicity in primary neurons was induced using hypoxia/hypoglycemia (H/H), veratridine (10microM), staurosporine (1microM) or glutamate (100microM), which resulted in 72%, 67%, 75% and 66% neuronal injury, respectively. 3-Aminopyridine-2-carboxaldehyde thiosemicarbazone (PAN-811; 10microM; Panacea Pharmaceuticals, Gaithersburg, MD) pretreatment for 24 h provided maximal neuroprotection of 89%, 42%, 47% and 89% against these toxicities, respectively. Glutamate or H/H treatment of cells increased cytosolic cytochrome c levels, which was blocked by pretreatment of cells with PAN-811. Pretreatment of neurons with PAN-811 produced a time-dependent increase in the protein level of Bcl-2, which was evident even after glutamate or H/H treatments. An up-regulation in the expression of the p53 and Bax genes was also observed following exposure to these neurotoxic insults; however, this increase was not suppressed by PAN-811 pretreatment. Functional inhibition of Bcl-2 by HA14-1 reduced the neuroprotective efficacy of PAN-811. PAN-811 treatment also abolished glutamate or H/H-mediated internucleosomal DNA fragmentation.

Lower standing systolic blood pressure as a predictor of falls in the elderly: a community-based prospective study.

OBJECTIVES: We investigated prospectively the relationships among falls, physical balance, and standing and supine blood pressure (BP) in elderly persons. BACKGROUND: Falls occur often and adversely affect the activities of daily living in the elderly; however, their relationship to BP has not been clarified thoroughly. METHODS: A total of 266 community-dwelling elderly persons age 65 years or over (123 men and 143 women, mean age of 76 years) were selected from among residents of Coop City, Bronx, New York. Balance was evaluated at baseline using computerized dynamic posturography (DPG). During a one-year follow-up, we collected information on subsequent falls on a monthly basis by postcard and telephone follow-up. RESULTS: One or more falls occurred in 60 subjects (22%) during the one-year follow-up. Women fell more frequently than men (28% vs. 16%, p < 0.03), and fallers were younger than nonfallers. Fallers (n = 60) had lower systolic BP (SBP) levels when compared with nonfallers (n = 206) (128 +/- 17 vs. 137 +/- 22 mm Hg for standing, p < 0.006; 137 +/- 16 vs. 144 +/- 22 mm Hg for lying, p < 0.02), whereas diastolic BP was not related to falls. Falls occurred 2.8 times more often in the lower BP subgroup (<140 mm Hg for standing SBP) than in the higher BP subgroup (> or =140 mm Hg, p < 0.0003), and gender-related differences were observed (p = 0.006): 3.4 times for women (p < 0.0001) versus 1.9 times for men (p = 0.30). Loss of balance, as detected by DPG, did not predict future falls and was also not associated with baseline BP levels. Multiple logistic regression analysis demonstrated that female gender (relative risk [RR] = 2.1, p = 0.02), history of falls (RR = 2.5, p = 0.008) and lower standing SBP level (RR = 0.78 for 10 mm Hg increase, p = 0.005) were independent predictors of falls during one year of follow-up. CONCLUSIONS: Lower standing SBP, even within normotensive ranges, was an independent predictor of falls in the community-dwelling elderly. Elderly women with a history of falls and with lower SBP levels should have more attention paid to the prevention of falls and related accidents.

Reliability and responsiveness of two physical performance measures examined in the context of a functional training intervention.

BACKGROUND AND PURPOSE: The reliability and responsiveness of 2 physical performance measures were assessed in this nonrandomized, controlled pilot exercise intervention. SUBJECTS: Forty-five older individuals with mobility impairment (mean age=77.9 years, SD=5.9, range=70-92) were sequentially assigned to participate in an exercise program (intervention group) or to a control group. METHODS: The intervention group performed exercise 3 times a week for 12 weeks that targeted muscle force, endurance, balance, and flexibility. Outcome measures were the 8-item Physical Performance Test (PPT-8) and the 6-minute walk test. Test-retest reliability and responsiveness indexes were determined for both tests; interrater reliability was measured for the PPT-8. RESULTS: The intraclass correlation coefficient for interrater reliability for the PPT-8 was. 96. Intraclass correlation coefficients for test-retest reliability were.88 for the PPT-8 and.93 for the 6-minute walk test. The intervention group improved 2.4 points and the control group improved 0.7 point on the PPT-8, as compared with baseline measurements. There was no change in 6-minute walk test distance in the intervention group when compared with the control group. The responsiveness index was.8 for the PPT-8 and.6 for the 6-minute walk test. CONCLUSION AND DISCUSSION: Measurements for both the PPT-8 and the 6-minute walk test appeared to be highly reliable. The PPT-8 was more responsive than the 6-minute walk test to change in performance expected with this functional training intervention.

Stance perturbation-evoked potentials in old people with poor gait and balance.

OBJECTIVE: Cerebral cortical potentials can be evoked by stance perturbation, and there is speculation that they represent the activation of supraspinal centers in preparation for the control and coordination of motor movements that maintain balance. We sought to determine if these potentials differed in old people at risk of falls. METHODS: Cortical potentials were generated by the sudden forward translation of a weight-bearing platform in 8 healthy young subjects and in 33 old subjects stratified by their functional capacity. Dependent measures were compared with non-parametric tests of significance. RESULTS: Perturbing the stance of young subjects produced a biphasic scalp potential centered at the vertex with an early positive peak at 60 ms and a larger, 45 microV, negative peak at 123 ms. In old subjects the response was delayed and the vertex-negative component was smaller and bifid. The interval between the two components of the negative peak was prolonged in a subgroup of old subjects with reduced mobility. CONCLUSIONS: Delays in sensory conduction may play a role in subsequent maladaptive motor responses to stance perturbation that can result in falls and injury in old people.

A possible chemical explanation for the events associated with the death of Gloria Ramirez at Riverside General Hospital.

The events associated with the death of Gloria Ramirez at Riverside General Hospital on 19 February 1994 have been portrayed as a major medical mystery. A potential chemical explanation for this incident has been developed. The hypothetical scenario depends upon the oxidation of a common solvent, dimethyl sulfoxide, through dimethyl sulfone to dimethyl sulfate. The latter compound is a volatile and highly toxic agent that can be quite hazardous to humans in small amounts. It is also environmentally nonpersistent. Much of the mystery surrounding the circumstances at the hospital may be explainable if this postulated metabolic pathway took place at the time of the emergency room incident. Although dimethyl sulfate was not detected in any analyses pertinent to this event, there are plausible scientific explanations to account for that fact. The sulfate anion, a hydrolysis product of dimethyl sulfate, was measured at an appreciably elevated concentration in Ramirez' blood. The descriptions of the symptoms of the hospital-staff victims appear quite consistent with dimethyl sulfate exposures. This paper attempts to make some sense of the reported data and eyewitness accounts, and perhaps provide new insight for any future research that could further explain this reported occurrence of toxic exposure.

Chemical and elemental comparison of two formulations of oleoresin capsicum.

In-custody deaths following the application of pepper spray weaponry by law enforcement personnel have increased in California over the last few years. Oleoresin capsicum (OC), an oily extract of hot peppers, is the active ingredient in the spray, but little detailed information on product mixtures is available. Since OC extracts contain a multitude of natural compounds at irregular concentrations, there could be considerable, variation in overall chemical composition among the different formulations of both 'natural' and 'synthetic' OC preparations. This was confirmed by organic and inorganic analyses performed on OC sprays produced by two manufacturers licensed for distribution within the state of California. The results indicated that the differences could lead to considerable inconsistency in weapon effectiveness, and suggested that more comprehensive studies are warranted.

Balance and strength training in older adults: intervention gains and Tai Chi maintenance.

OBJECTIVE: To determine the effect on balance and strength of 3 months of intensive balance and/or weight training followed by 6 months of low intensity Tai Chi training for maintenance of gains. DESIGN: Randomized control intervention. Four groups in 2 x 2 design: Control, Balance, Strength, Balance + Strength, using blinded testers. SETTING: Exercise and balance laboratory at University of Connecticut Health Center. PARTICIPANTS: Subjects were 110 healthy community dwellers (mean age 80) who were free of dementia, neurological disease, and serious cardiovascular or musculoskeletal conditions. INTERVENTIONS: Short-term training (3 months) occurred 3 times/week (45 minutes Balance and Strength, 90 minutes Balance + Strength). Balance training included equilibrium control exercises of firm and foam surfaces and center-of-pressure biofeedback. Strengthening consisted of lower extremity weight-lifting. All subjects than received long-term group Tai Chi instruction (6 months, 1 hour, 1 time/week). MEASUREMENTS: Losses of balance during Sensory Organization Testing (LOB), single stance time (SST), voluntary limits of stability (FBOS), summed isokinetic torque of eight lower extremity movements (ISOK), and usual gait velocity (GVU). RESULTS AND CONCLUSIONS: Balance training meaningfully improved all balance measures by restoring performance to a level analogous to an individual 3 to 10 years younger: LOB = -2.0 +/- 0.3 (adjusted paired differences, P < .005 ANOVA); SST = 7.0 +/- 1.2 sec; and FBOS = 9.0 +/- 2.0% of foot length (P < .05). Strengthening increased ISOK by 1.1 +/- 0.1 Nm kg-1 (P < .005). There was no interaction between balance and strength training. Significant gains persisted after 6 months of Tai Chi, although there was some decrement.

Strength is a major factor in balance, gait, and the occurrence of falls.

We studied the effects of lower extremity strength as well as gait and balance on the occurrence of falls in nursing home residents. Nursing home residents with a history of falls had less than half of the knee and ankle strength of nonfalling subjects residing in the same home. The differences were more prominent at the ankle than the knee, and were most pronounced in the ankle dorsiflexors, where they were one-tenth that of controls. Also of note was the fact that this same group of fallers had slowed gait velocity (58% of control) as well as an impaired response to postural perturbation as determined on the Postural Stress Test (55% of control). In a recently completed study we measured strength as balance (EquiTest balance platform) of community-dwelling subjects. The occurrence of loss of balance during the sensory organization test was correlated with diminished lower extremity (Pearson R = -.36, p = .001) as well as ankle dorsi and plantar flexion moments (Pearson R = -.37, p = .001). Using a logistic regression model, we demonstrated an independent effect of strength on the odds ratio of an SOT-LOB; for each newtonmeter per kg increase in strength there was a 20% decrease (95% CI, .74-87) in the odds ratio. The data from both nursing home and community-dwelling subjects indicate a strong relationship of lower extremity strength to balance and gait. The nursing home studies demonstrated an association between these functions and the occurrence of falls.(ABSTRACT TRUNCATED AT 250 WORDS)

Dynamic balance in older persons: effects of reduced visual and proprioceptive input.

BACKGROUND: Understanding the motor and sensory contributions to balance function in older persons is important in order to design effective interventions to prevent falls and loss of mobility. We tested the contributions of visual and proprioceptive input, muscle strength, and age to balance performance in 110 subjects free of clinical evidence of neurologic disease (mean age 80 years). METHODS: The EquiTest Sensory Organization Test was used to present sequential conditions that altered sensory input in a 2 (tactile/proprioceptive input) x 3 (visual input) design. Center of force and shear force data were recorded from the platform; the primary outcomes were loss of balance (LOB) and anterior-posterior center of force (COF) displacement as a proportion of foot length (COFD). RESULTS: Subjects had 3.5 +/- 2.7 LOB during testing. Reduction of vision input increased the odds ratio of a LOB 5.7 fold (3.9, 8.4, 95% CI) in unstable surface conditions, but adaptation with repeated trials was substantial. The adjusted odds ratio of a LOB was 0.65 and 0.45 in the second and third trials, respectively, compared to the first. Muscle strength and age were independent predictors of LOB. Gait velocity, single stance balance, and reported difficulty with ambulation, mobility, and instrumental activities of daily living were also associated with LOB. CONCLUSIONS: Performance on clinical tests of strength, balance, and gait was associated with performance on a challenging balance test which reduced sensory input and effectiveness of motor responses. Adaptation to challenging balance conditions was substantial in this group of older subjects, but was diminished in the oldest subjects.

Comprehensive forensic analyses of debris from the fatal explosion of a "cold fusion" electrochemical cell.

Selected components of explosion debris from the SRI International incident of January 2, 1992 were subjected to forensic analyses to elucidate potential causes of, or contributing factors to, the explosion. Interrogation of the debris encompassed nuclear, chemical, physical, and materials investigations. Nuclear studies for the determination of tritium and neutron-activation products in stainless steel and brass were conducted. No evidence for signature species indicative of orthodox nuclear events was detected. The inorganic and particulate analyses were likewise negative with respect to residues of unexpected chemical species. Such target compounds included conventional explosives, accelerants, propellants, or any exceptional industrial chemicals. Materials characterization identified the type of stainless steel used in the manufacture of the electrolytic cell as one relatively high in Mo concentration, probably type 316. Metallurgical analyses of the cell vessel wall and its detached base provided no evidence of corrosion or hydrogen embrittlement, leaving only ductile failure of the weld as contributing to the incident. The weld was found to have missed the center-line of the step joint, and the average penetration of the weld was measured to be 54%. The GC-MS analyses of trace organic components in the explosion debris provided a most interesting result. Although no evidence of organic explosives, oxidizers, or other unusual compounds was detected, the presence of an organic oil in the interior of the electrochemical cell was established. It is likely that the source of this oil was lubricating fluid from machining the metal cell components.(ABSTRACT TRUNCATED AT 250 WORDS)

Clinical application of the forearm tourniquet.

We undertook this prospective study of 148 consecutive hand and wrist procedures to evaluate the safety and efficacy of the forearm tourniquet. Patients were assigned to three groups based on tourniquet pressure used, ranging from 50 to 125 mmHg above systolic blood pressure. The forearm tourniquet was well tolerated by all patients. No patient had neurologic complications. Minimal intraoperative bleeding occurred when the pressure was greater than 75 mmHg above systolic blood pressure. The forearm tourniquet is a safe and effective means of providing a bloodless field for wrist and hand surgery.

Effects of resistive and balance exercises on isokinetic strength in older persons.

OBJECTIVE: To determine the safety and efficacy of 3 months of resistive training of multiple lower extremity muscle groups compared with balance training in persons over 75 years. DESIGN: Randomized 3-month clinical trial. Subjects (n = 110, mean age 80) were randomized to 4 groups in a 2 x 2 design (control, resistive, balance, combined resistive/balance). INTERVENTIONS: Resistive training involved knee extension and flexion, hip abduction and extension, and plantar and dorsiflexion using simple resistive machines and sandbags. Balance training consisted of exercises to improve postural control. The control group attended 5 health-related discussion sessions. MEASUREMENTS: Summed isokinetic moments (N m) of 8 leg movements: hip, knee and ankle flexion/extension, and hip abduction/adduction. Secondary outcomes were gait velocity and chair rise time. MAIN RESULTS: Summed peak moment increased in both resistive exercise-trained groups (13% increase in the resistive group and 21% in the combined training group, P < 0.001). The effect of resistance training was significant (MANOVA F = 21.1, P < 0.001), but balance training did not improve strength, and there was no interaction (positive or negative) between balance and resistive training. Maximal gait velocity and chair rise time did not improve. Eleven subjects (20%) had musculoskeletal complaints related to resistive training, but all were able to complete the program with modifications. CONCLUSION: Resistive training using simple equipment is an effective and acceptable method to increase overall leg strength in older persons. Resistive or balance training did not improve maximal gait velocity or chair rise time in this sample of relatively healthy older persons.

Gender differences in the balance of healthy elderly as demonstrated by dynamic posturography.

BACKGROUND: Prior studies indicate that older women fall more often than men although there is no evidence of gender-based balance differences. Using a force platform, we measured the effects of restricted sensory input and support surface movement to detect gender differences in balance. METHODS: Healthy, elderly community dwellers (N = 234, mean age = 76 +/- 5 years, 52% female) were administered the following perturbations on the balance platform: The platform and/or visual surround were fixed or tilted proportionally to the subject's sway with the eyes open or closed, forward or backward horizontal translations, and toes-up and toes-down rotations. RESULTS: Gender-based balance differences were not present during quiet standing, or when the support surface or visual input were manipulated separately. Women swayed and lost their balance more than men when the surface was sway-referenced while vision was compromised, but by the third trial their sway control was comparable to the men. Women also initially lost their balance more frequently than men during toes-up and -down rotations, and compared to men continued to lose their balance more often during repeated toes-up rotations. Finally, women developed less angular momentum than men in response to forward platform rotations. DISCUSSION: Elderly women show impairments of balance when simultaneously deprived of visual and somatosensory inputs or during a backwards destabilization. Since there is little evidence for a CNS source for such gender differences, biomechanical origins (e.g., dorsiflexion strength and range of motion) are a more likely cause. Limited postural control of women under conditions stressing balance may explain their greater frequency of falling.

Ejection of the proximal scaphoid in a trans-scaphoid perilunate fracture dislocation. A case report.

Trans-scaphoid perilunate fracture dislocations are difficult injuries to treat. Described herein is the unusual case of a trans-scaphoid perilunate fracture dislocation in which the proximal scaphoid fragment was ejected into the volar aspect of the distal forearm. The significant displacement of the proximal fragment represented complete devascularization of this piece. This unique fracture raised difficult management issues in the treatment of this injury. A primary proximal row carpectomy was performed, with a successful clinical result.

Altered sensory function and balance in older persons.

The effects of three different visual inputs (eyes open, eyes closed, and inaccurate) while standing on an unstable surface (respectively, UEO, UEC, UI) were compared in a group of healthy elderly community dwellers (N = 239; mean age 76) and young (N = 34; mean age 34) adults. Subjects with medical factors known to affect balance were excluded. Body sway and loss of balance measured dynamic force platform (EquiTest, NeuroCom International). Visual and somatosensory inputs were rendered inaccurate through tilting of the standing surface and/or the visual surround proportional to the subject's angle of sway (sway-referencing). The elderly group had significantly more difficulty balancing during UEC and UI, and a larger proportion lost their balance during UI. These findings are compared to those of other dynamic posturography studies in which sensory inputs were controlled. Issues of age, frailty, screening, and test protocol are addressed in order to account for differences in results among studies.