Abnormal tau induces cognitive impairment through two different mechanisms: synaptic dysfunction and neuronal loss.

The hyperphosphorylated microtubule-associated protein tau is present in several neurodegenerative diseases, although the causal relationship remains elusive. Few mouse models used to study Alzheimer-like dementia target tau phosphorylation. We created an inducible pseudophosphorylated tau (Pathological Human Tau, PH-Tau) mouse model to study the effect of conformationally modified tau in vivo. Leaky expression resulted in two levels of PH-Tau: low basal level and higher upon induction (4% and 14% of the endogenous tau, respectively). Unexpectedly, low PH-Tau resulted in significant cognitive deficits, decrease in the number of synapses (seen by EM in the CA1 region), reduction of synaptic proteins, and localization to the nucleus. Induction of PH-Tau triggered neuronal death (60% in CA3), astrocytosis, and loss of the processes in CA1. These findings suggest, that phosphorylated tau is sufficient to induce neurodegeneration and that two different mechanisms can induce cognitive impairment depending on the levels of PH-Tau expression.

N-cadherin/FGFR promotes metastasis through epithelial-to-mesenchymal transition and stem/progenitor cell-like properties.

N-cadherin and HER2/neu were found to be co-expressed in invasive breast carcinomas. To test the contribution of N-cadherin and HER2 in mammary tumor metastasis, we targeted N-cadherin expression in the mammary epithelium of the MMTV-Neu mouse. In the context of ErbB2/Neu, N-cadherin stimulated carcinoma cell invasion, proliferation and metastasis. N-cadherin caused fibroblast growth factor receptor (FGFR) upmodulation, resulting in epithelial-to-mesenchymal transition (EMT) and stem/progenitor like properties, involving Snail and Slug upregulation, mammosphere formation and aldehyde dehydrogenase activity. N-cadherin potentiation of the FGFR stimulated extracellular signal regulated kinase (ERK) and protein kinase B (AKT) phosphorylation resulting in differential effects on metastasis. Although ERK inhibition suppressed cyclin D1 expression, cell proliferation and stem/progenitor cell properties, it did not affect invasion or EMT. Conversely, AKT inhibition suppressed invasion through Akt 2 attenuation, and EMT through Snail inhibition, but had no effect on cyclin D1 expression, cell proliferation or mammosphere formation. These findings suggest N-cadherin/FGFR has a pivotal role in promoting metastasis through differential regulation of ERK and AKT, and underscore the potential for targeting the FGFR in advanced ErbB2-amplified breast tumors.

N-cadherin regulates mammary tumor cell migration through Akt3 suppression.

N-cadherin is a cell-cell adhesion molecule that plays a role in breast cancer metastasis. Here, we show that in vivo expression of N-cadherin in the PyMT mouse model, which enhances mammary tumor metastasis, results in selective inhibition of Akt3 expression and phosphorylation. Similarly, exogenous expression of N-cadherin in PyMT or MCF-7 mammary tumor cells enhanced cell motility and caused a dramatic reduction in Akt3 expression and phosphorylation. Moreover, knockdown of Akt3 in PyMT tumor cells increased cell motility and disrupted mammary morphogenesis, but had no effect on cell proliferation. Conversely, overexpression of wild-type Akt3 in PyMT-N-cadherin cells inhibited cell motility promoted by N-cadherin. Taken altogether, these findings demonstrate that N-cadherin suppresses Akt3 to promote cell motility and highlight the intricate regulation of Akt isoforms by N-cadherin during metastasis.

p21CIP1 mediates reciprocal switching between proliferation and invasion during metastasis.

Cell proliferation and invasion are critical for malignant progression, yet how these processes relate to each other and whether they regulate one another during metastasis is unknown. We show that invasiveness of breast cancer cells is associated with growth arrest due to p21CIP1 upregulation. Knockdown of p21CIP1 increases cell proliferation and suppresses invasion. Since p21CIP1 acts to inhibit cyclin E during cell-cycle progression, we demonstrated that a constitutively active form of cyclin E had similar effects to p21CIP1 inhibition resulting in enhanced cell growth and suppressed invasiveness. We tested these findings in vivo in the Polyoma middle T mammary tumor model in which p21CIP1 was deleted. p21CIP1 knockout mice exhibited dramatic suppression of metastasis, independent of tumor growth, which was rescued by p21CIP1. Metastasis suppression by p21CIP1 ablation was associated with striking cytoskeletal reorganization leading to a non-invasive and highly proliferative state. Thus, p21CIP1 regulates metastasis by mediating reciprocal switching between invasion and proliferation.

The presynaptic particle web: ultrastructure, composition, dissolution, and reconstitution.

We report the purification of a presynaptic "particle web" consisting of approximately 50 nm pyramidally shaped particles interconnected by approximately 100 nm spaced fibrils. This is the "presynaptic grid" described in early EM studies. It is completely soluble above pH 8, but reconstitutes after dialysis against pH 6. Interestingly, reconstituted particles orient and bind PSDs asymmetrically. Mass spectrometry of purified web components reveals major proteins involved in the exocytosis of synaptic vesicles and in membrane retrieval. Our data support the idea that the CNS synaptic junction is organized by transmembrane adhesion molecules interlinked in the synaptic cleft, connected via their intracytoplasmic domains to the presynaptic web on one side and to the postsynaptic density on the other. The CNS synaptic junction may therefore be conceptualized as a complicated macromolecular scaffold that isostatically bridges two closely aligned plasma membranes.

Comparison of physiological responses to open water kayaking and kayak ergometry.

This study compared the physiological responses of simulated kayaking on a K1 ERGO kayak ergometer with open water paddling. Nine well-trained male kayakers (VO2peak 4.27 +/- 0.58 L x min(-1), age 24 +/- 4 yr, mass 77.3 +/- 6.4 kg, height 179.5 +/- 5.3 cm; [mean +/- SD]) performed two 4 min exercise bouts on open water (OW) and on an air braked kayak ergometer (Erg). During exercise, expired air and heart rate (HR) were continuously measured. The distance covered during OW (992 +/- 47.1 m) was highly correlated (r2 = 0.86) with the total work performed in Erg (47.64 +/- 7.67 kJ). There were no differences between trials for oxygen uptake, carbon dioxide production or estimated carbohydrate oxidation. However, during OW, minute ventilation was significantly higher at 60 and 90 s (104.2 +/- 16.4 vs. 92.6 +/- 20.4 L x min(-1) and 120.5 +/- 15.8 vs. 111.7 +/- 17.6 L x min(-1) for 60 and 90 s, respectively, p < 0.05), and HR was higher in OW during the first minute (120 +/- 20 vs. 104 +/- 19 beats x min(-1), 164 +/- 8 vs. 147 +/- 18 beats x min(-1) and 178 +/- 6 vs. 170 +/- 7 beats x min(-1) for 0, 30, and 60 s, respectively, p < 0.05). There were no differences in peak VO2 between OW and Erg (4.10 +/- 0.49 vs. 4.09 +/- 0.53 L x min(-1), respectively) nor in post-exercise blood (lactate) (6.43 +/- 1.47 vs. 6.59 +/- 0.99 mmol x L(-1), respectively). We conclude that the K1 ERGO accurately simulates the physiological demands of short-term, high-intensity kayaking.

Molecular modification of N-cadherin in response to synaptic activity.

The relationship between adhesive interactions across the synaptic cleft and synaptic function has remained elusive. At certain CNS synapses, pre- to postsynaptic adhesion is mediated at least in part by neural (N-) cadherin. Here, we demonstrate that upon depolarization of hippocampal neurons in culture by K+ treatment, or application of NMDA or alpha-latrotoxin, synaptic N-cadherin dimerizes and becomes markedly protease resistant. These properties are indices of strong, stable, enhanced cadherin-mediated intercellular adhesion. N-cadherin retained protease resistance for at least 2 hr after recovery, while other surface molecules, including other cadherins, were completely degraded. The acquisition of protease resistance and dimerization of N-cadherin is not dependent on new protein synthesis, nor is it accompanied by internalization of N-cadherin. By immunocytochemistry, we found that high K+ selectively induces surface dispersion of N-cadherin, which, after recovery, returns to synaptic puncta. N-cadherin dispersion under K+ treatment parallels the rapid expansion of the presynaptic membrane consequent to the massive vesicle fusion that occurs with this type of depolarization. In contrast, with NMDA application, N-cadherin does not disperse but does acquire enhanced protease resistance and dimerizes. Our data strongly suggest that synaptic adhesion is dynamically and locally controlled, and modulated by synaptic activity.

Exogenous expression of N-cadherin in breast cancer cells induces cell migration, invasion, and metastasis.

E- and N-cadherin are calcium-dependent cell adhesion molecules that mediate cell-cell adhesion and also modulate cell migration and tumor invasiveness. The loss of E-cadherin-mediated adhesion has been shown to play an important role in the transition of epithelial tumors from a benign to an invasive state. However, recent evidence indicates that another member of the cadherin family, N-cadherin, is expressed in highly invasive tumor cell lines that lacked E-cadherin expression. These findings have raised the possibility that N-cadherin contributes to the invasive phenotype. To determine whether N-cadherin promotes invasion and metastasis, we transfected a weakly metastatic and E-cadherin-expressing breast cancer cell line, MCF-7, with N-cadherin and analyzed the effects on cell migration, invasion, and metastasis. Transfected cells expressed both E- and N-cadherin and exhibited homotypic cell adhesion from both molecules. In vitro, N-cadherin-expressing cells migrated more efficiently, showed an increased invasion of Matrigel, and adhered more efficiently to monolayers of endothelial cells. All cells produced low levels of the matrix metalloproteinase MMP-9, which was dramatically upregulated by treatment with FGF-2 only in N-cadherin-expressing cells. Migration and invasion of Matrigel were also greatly enhanced by this treatment. When injected into the mammary fat pad of nude mice, N-cadherin-expressing cells, but not control MCF-7 cells, metastasized widely to the liver, pancreas, salivary gland, omentum, lung, lymph nodes, and lumbar spinal muscle. The expression of both E- and N-cadherin was maintained both in the primary tumors and metastatic lesions. These results demonstrate that N-cadherin promotes motility, invasion, and metastasis even in the presence of the normally suppressive E-cadherin. The increase in MMP-9 production by N-cadherin-expressing cells in response to a growth factor may endow them with a greater ability to penetrate matrix protein barriers, while the increase in their adherence to endothelium may improve their ability to enter and exit the vasculature, two properties that may be responsible for metastasis of N-cadherin-expressing cells.

Functional cis-heterodimers of N- and R-cadherins.

Classical cadherins form parallel cis-dimers that emanate from a single cell surface. It is thought that the cis-dimeric form is active in cell-cell adhesion, whereas cadherin monomers are likely to be inactive. Currently, cis-dimers have been shown to exist only between cadherins of the same type. Here, we show the specific formation of cis-heterodimers between N- and R-cadherins. E-cadherin cannot participate in these complexes. Cells coexpressing N- and R-cadherins show homophilic adhesion in which these proteins coassociate at cell-cell interfaces. We performed site- directed mutagenesis studies, the results of which support the strand dimer model for cis-dimerization. Furthermore, we show that when N- and R-cadherins are coexpressed in neurons in vitro, the two cadherins colocalize at certain neural synapses, implying biological relevance for these complexes. The present study provides a novel paradigm for cadherin interaction whereby selective cis-heterodimer formation may generate new functional units to mediate cell-cell adhesion.

Domains of tenascin involved in glioma migration.

Tenascin (TN) is an extracellular matrix protein found in areas of cell migration during development and expressed at high levels in migratory tumor cells. TN was previously shown to support the attachment and migration of glioma cells in culture. To determine the domains responsible for glioma migration and attachment, we produced recombinant fusion proteins that collectively span the majority of the molecule including its epidermal growth factor-like repeats, fibronectin type III repeats and fibrinogen domain. These domains were tested for their ability to support migration of C6 glioma cells in an aggregate migration assay. A recombinant fusion protein including fibronectin type III (FNIII) repeats 2-6 (TNfn2-6) was the only fragment found to promote migration of C6 glioma cells at levels similar to that promoted by intact TN. Evaluation of smaller segments and individual FNIII repeats revealed that TNfn3 promoted migration and attachment of glioma cells and TNfn6 promoted migration but not attachment. While TNfn3 and TNfn6 promoted migration individually, the presence of both TNfn3 and TNfn6 was required for migration on segments of the FNIII region that included TNfn5. TNfn5 inhibited migration in a dose dependent manner when mixed with TNfn3 and also promoted strong attachment and spreading of C6 glioma cells. TNfn3 and TNfn6 promote cell migration and may function cooperatively to overcome the inhibitory activity of TNfn5. Additional cell attachment studies suggested that both beta1 integrins and heparin may differentially influence the attachment of glioma cells to TN fragments. Together, these findings show that C6 glioma cells integrate their response upon binding to at least three domains within TN.

Developmental expression of two rat sialyltransferases that modify the neural cell adhesion molecule, N-CAM.

Polysialylation of the neural cell adhesion molecule (N-CAM) reduces the efficacy of N-CAM-mediated homophilic binding and is regulated both during development and in regions undergoing neurogenesis or remodeling in the adult. Hamster PST-1 (PST) and rat STX are two related sialytransferases that catalyze the polysialylation of N-CAM. We have isolated a cDNA clone for the rat homologue of PST and compared its amino acid and nucleotide sequence to that of rat STX. This analysis revealed regions of high sequence similarity corresponding to the enzymatic domains of the two molecules. Other regions of lower similarity were used to generate specific probes for in situ hybridization. The distribution of PST and STX mRNAs, polysialic acid, and N-CAM were analyzed at three developmental stages. PST and STX mRNAs were expressed abundantly throughout the nervous system at embryonic day 15 and postnatal day 4 and were coexpressed in most tissues examined. In the adult brain, STX expression was reduced relative to PST and expression of both mRNAs was restricted to subsets of cells in areas undergoing constant synaptic rearrangement including hippocampus and olfactory system. The results suggest that both PST and STX participate in the polysialylation of N-CAM in vivo and that their expression levels are dynamically controlled during development and regeneration.

Barrier precautions in trauma resuscitation: real-time analysis utilizing videotape review.

Blood-borne pathogens threaten all individuals involved in emergency health care. Despite recommendations by the Centers for Disease Control and the American College of Emergency Physicians, documented compliance with universal precautions in trauma resuscitation has been poor. The purpose of this study was to determine the factors that predispose to noncompliance with barrier precautions at a level I trauma center. Videotapes of trauma resuscitations performed during 1 month (n = 66) were reviewed. Full compliance with barrier precautions was documented in 89.1% of health care workers. Of the noncompliant health care workers, 50.7% were emergency department personnel and 47.8% were first responders to the trauma resuscitation area. Barrier precaution compliance improved from 62.5% to 91.8% with prenotification of patient arrival. Immediate access to barrier equipment is essential for all potential in-hospital first responders. Prehospital communication systems should be optimized to ensure prenotification.

Bronchoscopic guidance makes percutaneous tracheostomy a safe, cost-effective, and easy-to-teach procedure.

BACKGROUND: We wanted to assess the efficiency of instituting a modified technique of percutaneous tracheostomy (PET) with bronchoscopic guidance. METHODS: During a 10-month period 48 consecutive trauma patients requiring tracheostomy were divided between a standard tracheostomy control group (ST) and a PET group. All patients were followed prospectively. The hospital charges were reviewed retrospectively. RESULTS: Age, gender, body habitus, and principal diagnosis were similar in the 21 ST patients and the 27 PET patients. All STs and 15 of the PETs were performed in the operating room (OR), and the 12 remaining PETs were done in the intensive care unit (ICU). Four patients in the ST group and six in the PET group died. One of these deaths occurred in a patient in the PET group with severe adult respiratory distress syndrome. Procedure time was shorter for PET (16 versus 45 minutes, p < 0.0001). Junior residents performed more PETs than STs (33% versus 10%), and PET was considered "easier" to perform than ST (81% versus 47%). Hospital charges for PET in the ICU were $3400 less per patient compared with ST or PET in the OR. CONCLUSIONS: PET was performed easily and safely in the OR and at the ICU bedside. PET required one-third the time of ST. Bronchoscopic supervision of PET may have contributed to the small number of complications and the educational experience of junior residents. PET in the ICU can reduce hospital charges significantly and avoids transport of patients to the OR. PET is as safe as ST and should be considered the procedure of choice for an ICU patient requiring an elective tracheostomy.

Functional analysis of posttranslational cleavage products of the neuron-glia cell adhesion molecule, Ng-CAM.

Neuron-glia cell adhesion molecule (Ng-CAM) mediates cell adhesion between neurons homophilically and between neurons and glia heterophilically; it also promotes neurite outgrowth. In the chick brain, Ng-CAM is detected as glycoproteins of 190 and 210 kD (Ng-CAM200) with posttranslational cleavage products of 135 kD (F135, which contains most of the extracellular region) and 80 kD (F80, which includes the transmembrane and the cytoplasmic domains). To examine the functions of each of these components, we have expressed Ng-CAM200, F135, and F80 in murine L cells, and F135 and F80 as GST fusion proteins in the pGEX vector in bacteria. Appropriately transfected L cells expressed each of these proteins on their surfaces; F135 was also found in the media of cells transfected with Ng-CAM200 and F135. In addition to binding homophilically, cells transfected with Ng-CAM200 and F135 bound heterophilically to untransfected L cells, suggesting that there is a ligand for Ng-CAM on fibroblasts that may be related to the glial ligand. Detailed studies using the transfected cells and the fusion proteins indicated that both the homophilic and the heterophilic binding activities of Ng-CAM are localized in the F135 fragment of the molecule. The results also indicated that proteolytic cleavage of Ng-CAM200 is not required either for its expression on the cell surface or for cell adhesion and that there is an "anchor" for F135 on L cells (and presumably on neurons). In contrast to the cell binding results, the F80 but not the F135 fusion protein enhanced the outgrowth of neurites from dorsal root ganglion cells; this activity was associated with the FnIII repeats of F80. The observations that a protein corresponding to F135 contains the cell aggregation sites whereas one corresponding to the F80 has the ability to promote neurite outgrowth suggest that proteolytic cleavage may be an important event in regulating these Ng-CAM activities during embryonic development and neural regeneration.

Separate cell binding sites within cytotactin/tenascin differentially promote neurite outgrowth.

Cytotactin/tenascin (CT/TN) is an extracellular matrix protein that binds to a variety of cell types and that influences neurite outgrowth. It has a multidomain structure with regions homologous to epidermal growth factor (EGF)-like repeats, fibronectin (FN) type II repeats, and the beta and gamma chains of fibrinogen (fg). The current study demonstrates that a fusion protein corresponding to the sixth fibronectin type III repeat in CT/TN (CTfn6) supported cell attachment and promoted an increase in the number of cells with neurites in both central and peripheral neurons in tissue culture. The third fibronectin type III repeat, CTfn3, like intact CT/TN, supported attachment of peripheral neurons but not of central neurons and, while it caused an increase in neurite length, it did not increase the number of cells that sprouted neurites. When CTfn3 and CTfn6 were combined, an increase in both the number of cells sprouting neurites and in neurite length was observed for peripheral neurons that resembled their response to intact CT/TN. Cell attachment to CTfn6 was inhibited in the presence of function-blocking antibodies against beta 1 integrins. In contrast, the interaction with CTfn3 was not inhibited by antibodies to beta 1 integrins, but was inhibited by RGD-containing peptides. The results suggest that cell binding to CT/TN involves two different sites within the molecule and occurs via different receptors which may be differentially expressed on different neuronal cell types. The location of these sites within the whole molecule in the context of other adhesive and counteradhesive domains may modulate their influence on cellular responses such as cell attachment and neurite outgrowth.

Gluteal gunshot wounds: who warrants exploration?

It is difficult to determine which stable patients with gluteal gunshot wounds warrant exploration since 22% to 36% will have injuries requiring operative intervention. The ability of preoperative studies to identify major injuries was evaluated to determine which studies could accurately triage patients into a high-risk group that would warrant laparotomy and a low-risk group that could be managed with observation. The findings of abdominal tenderness or gross blood in the urine or rectum were each highly predictive of major injury. The determination of an extrapelvic versus transpelvic bullet trajectory allowed accurate triage of 94% of patients. Nearly 85% of patients with a transpelvic trajectory had injuries that required operative intervention. No patients with an extrapelvic trajectory required laparotomy. Given the density of vital structures above and below the peritoneum in the pelvis, we conclude that any patient with a transpelvic bullet trajectory warrants exploration.

Massive blood loss in trauma patients. The benefits and dangers of transfusion therapy.

In acutely injured patients, recognition of profound shock may be difficult initially. Ensuring adequate oxygenation, restoring intravascular volume, and controlling ongoing blood loss are key principles of treatment in these patients. Additionally, an appreciation for and recognition of the possible adverse consequences of massive transfusion (ie, hypothermia, coagulopathy, hypocalcemia, hyperkalemia, and hemolysis) enable physicians to prevent them or at least lessen their effects.

'Damage control': an approach for improved survival in exsanguinating penetrating abdominal injury.

Definitive laparotomy (DL) for penetrating abdominal wounding with combined vascular and visceral injury is a difficult surgical challenge. Physiologic derangements such as dilutional coagulopathy, hypothermia, and acidosis often preclude completion of the procedure. "Damage control" (DC), defined as initial control of hemorrhage and contamination followed by intraperitoneal packing and rapid closure, allows for resuscitation to normal physiology in the intensive care unit and subsequent definitive re-exploration. The purpose of the study was to compare the damage control technique with definitive laparotomy. Over a 3 1/2-year period, 46 patients with penetrating abdominal injuries required laparotomy and urgent transfusion of greater than 10 units packed red blood cells for exsanguination. Medical records were retrospectively reviewed for degree and pattern of injury, probability of survival, actual survival, transfusion requirements for the preoperative and postoperative phases, resuscitation and operative times, lowest perioperative temperature, pH, and HCO3. No significant differences were identified between 22 DL and 24 DC patients and actual survival rates were similar (55% DC vs. 58% DL). However, in a subset of 22 patients with major vascular injury and two or more visceral injuries (maximum injury subset), otherwise similar to the overall group, survival was markedly improved in patients treated with damage control (10 of 13, 77%*) vs. DLM (1 of 9, 11%) (Fisher's exact test, * p < 0.02). In preparation for return to the operating room, DC survivors averaged 8.4 units of packed red blood cells transfused and 10.3 units fresh frozen plasma over a mean ICU stay of 31.7 hours. Resolution of coagulopathy (mean prothrombin time/partial thromboplastin time 19.5/70.4 to 13.3/34.9), normalization of acid-base balance (mean pH/HCO3 7.37/20.6 to 7.42/24.2), and core rewarming (mean 33.2 degrees C to 37.7 degrees C) were achieved. All patients had gastrointestinal procedures at reoperation (mean operative time, 4.3 hours). We conclude that damage control is a promising approach for increased survival in exsanguinating patients with major vascular and multiple visceral penetrating abdominal injuries.

Global analysis of steady-state polarized fluorescence spectra using trilinear curve resolution.

Global analysis using trilinear curve resolution is described and shown to be a powerful method for the resolution of polarized fluorescence data arrays, in which the measured fluorescence intensity is a separable function of polarization orientation, excitation wavelength, and emission wavelength. This methodology is applicable to mixtures the components of which have linearly independent excitation and emission spectra and distinct anisotropies. Normalized excitation and emission spectra of individual components can be uniquely determined without prior assumptions concerning spectral shapes (e.g., sum of Gaussians) and without the uncertainties inherent in bilinear techniques such as principal component analysis or factor analysis. The normalized excitation and emission vectors are combined with the total absorption spectrum of the multicomponent mixture to compute absolute absorption and emission spectra. The precision of this methodology is evaluated as a function of noise, overlap, relative intensity, and anisotropy difference between components using simulated mixtures of the DNA bases. The ability of this method to extract individual spectra from steady-state fluorescence data arrays is illustrated for mixtures containing two and three components.