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The primary outcome variables were mood scores, anxiety scores, and thalamic GABA levels. Continuous measures were summarized by means_standard deviations; withingroup comparisons were performed using paired t-tests, while between-group comparisons were performed using two-sample t-tests. Discrete measures were summarized by raw counts for numerators and denominators, as well as the associated percentages, and were compared by Fisher’s exact test due to the limited sample size. Linear regression analysis was used to quantify the association between the primary outcome variables and potential predictor variables. In order to take into account within-subject correlations arising from repeated longitudinal measurements, generalized estimated equations (GEEs) were used to analyze within-group trends in mood and anxiety scores, as well as to perform betweengroup analyses. All hypothesis tests were two-tailed and conducted at the aј0.05 significance level. Confidence intervals were two-sided and were constructed with 95% confidence. Stata 10.0 (College Station, TX) was used for analysis.

Results
Demographics and study participation
Thirty-four subjects completed the study: 19 in the yoga group and 15 in the walking group. There was no significant difference between groups for demographic or descriptive variables except for height, which although statistically significant due to a relatively small standard deviation was clinically not significant. There was no difference in demographics between study completers and dropouts, with dropouts equally divided between interventions. The means for the weekly PAR METs during the 12-week intervention showed the walking group to have a significantly greater level of activity outside the intervention than the yoga group ( pј0.02); however, there was no difference between groups in activity levels on the week before Imaging Session II. Out of 36 sessions, each group attended about two thirds, with the yoga group reporting about one session a week at home.
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Analysis of mood and anxiety scales and GABA levels The following analyses were done with statistically significant findings reported in the tables: (1) a GEE model for changes in mood and anxiety scores for each group at weeks 0, 4, 8, and 12; (2) tonic changes in GABA levels were assessed over the course of the intervention by subtracting Scan 1 from Scan 2 values, while acute changes associated with the intrascan session were assessed by subtracting Scan 2 from Scan 3 values; (3) tonic and acute changes in GABA levels; (4) correlations of mood and anxiety scores with GABA levels for each scan; (5) correlations of tonic (Scan 2–1) and acute (Scan 3–2) changes in mood and anxiety scores with tonic and acute changes in GABA levels. In the three ‘‘positive’’ subscales of EIFI (Positive Engagement, Revitalization, and Tranquility), an increase in score indicates improved mood. In the two ‘‘negative’’ scales, the STAI-State and EIFI-Physical Exhaustion, an increase in score indicates increased anxiety and physical exhaustion, respectively. Inverse associations (-beta), with the negative scales, indicate decreased anxiety in the within-group analysis and greater decrease in anxiety for the yoga group in the between-group analysis.

Certified Iyengar yoga instructors taught the yoga interventions, which were monitored by the Principal Investigator to ensure consistency in presentation of weekly posture sequences. Written lists of the weekly sequences and pictures of the postures were given to the subjects. After 4 weeks of instruction, subjects were encouraged to practice at home. The intrascan yoga sequence was taught in class and monitored by research staff during Imaging Session II. The structure of the walking intervention was designed to be similar to that of the yoga intervention, with weekly group sessions in which subjects walked around the gym perimeter at 2.5mph for 60 minutes. The intrascan walking session was done on a treadmill set to 2.5mph with 0 incline. This design controlled for group effects and interaction time with research staff.

Imaging Subjects were scanned on a 4-Tesla full-body MR scanner (Varian/UnityInova, Varian Inc., Palo Alto, CA) at Mclean Hospital in Belmont, MA. Scout images confirmed optimal positioning. After global shimming on unsuppressed water, T1-weighted anatomical images were taken in sagittal and axial planes [echo time (TE)/repetition time (TR)ј6.2 seconds/ 11.4 milliseconds, field-of-viewј22_22_8 cm(sagittal) and 22_22_16cm (axial), readout durationј4ms, receive bandwidthј_32 kHz, in-plane matrix sizeј128_256_16 (sagittal) and 256_256_64 (axial), in-plane resolutionј 0.94_1.9mm (sagittal) and 0.94_0.94mm (axial), readout pointsј512, slice-thicknessј2.5mm, flip-angleј118]. In our previous study, a post-hoc regional analysis that used multivoxel spectroscopic imaging showed that the greatest increase in GABA levels after the yoga intervention was in the thalamus.8 The selection of the left thalamus was based on evidence that the left side has greater parasympathetic innervations and that GABA levels are lower in the left thalamus in post-traumatic stress disorder subjects. For this study, an algorithm was developed to position a 2_2_3- cm voxel over the left thalamus. Proton spectroscopy implemented a MEGAPRESS [MEscher-GArwood Point- Resolved Echo Spectroscopy Sequence] difference-editing sequence specifically tuned for GABA.24 Manual voxel shimming yielded global water-line widths ranging from 8 to 15Hz. The MEGAPRESS sequence collected 68-millisecond echo-time spectra in an interleaved fashion where the GABA editing pulse was applied on every second transient. Additional MEGAPRESS acquisition parameters were: TRј2 seconds, spectral-bandwidthј2 kHz, readout-durationј512 milliseconds, Number of Excitations (NEX)ј384, and total scan durationј13 minutes.

In order to quantify GABA, the difference-edited spectra were processed and then fitted with LCModel using basis sets acquired at 4 T. A separate LCModel template was used to fit the unedited 68-milisecond subspectrum to obtain creatine (Cr). All fitted metabolite areas were normalized to the fitted Cr resonance from the 68-millisecond subspectrum. One (1) spectrum from the MEGAPRESS acquisition in the thalamus was excluded from analysis due to low signal-to-noise. GABA/Cr ratios are referred to as GABA levels. In order to ascertain the gray and white matter contribution to each voxel, the axial T1-weighted images were segmented into gray matter, white matter, and cerebrospinal fluid compartments using the commercial software package FSL 4.1 (FMRIB Software Library; Analysis Group, FMRIB; Oxford, UK).

There is a large body of research on the beneficial effects of exercise on depression and anxiety. The results of exercise as a treatment for mild to moderate depression compare favorably to psychotherapy and pharmacologic treatment, supporting the contention that a behavioral intervention can have an effect similar to a pharmacologic intervention on mood.

This study extends the findings of self-reported mood changes by exploring a possible mechanism: changes in thalamic GABA levels measured on MRS. This study was designed to correlate changes in mood, anxiety, and brain GABA levels, and to determine whether such changes are specific to a practice of yoga postures or whether they occur in a metabolically matched walking intervention. We hypothesize that improvement in mood scores correlate positively with GABA levels, while anxiety scores will correlate negatively with GABA levels.

Materials and Methods Subjects were recruited from the community by newspaper ads, flyers, and the Internet. Screening interviews and written informed consent were obtained at Boston University School of Medicine General Clinical Research Unit. Eligible subjects were randomized in permuted blocks (nј4) to a 12- week intervention of either Iyengar yoga or walking for three 60-minute sessions per week, with a maximum of 36 sessions. All subjects had three MRS scans: Scan 1 at baseline; Scan 2 after their 12-week intervention; immediately after Scan 2, all subjects completed a 60-minute yoga or walking intervention, depending on group assignment, which was immediately followed by Scan 3. Participants were 18–45 years old with no current Axis I diagnosis. Nonpsychoactive medications were allowed if the subject had been on a stable dose for at least 1 month with no anticipated changes during the study. The following items were exclusionary: any yoga practice in the previous 3 months, or a lifetime history of one yoga session/week for _4 weeks; current participation in psychotherapy, prayer groups, or any mind–body disciplines; a neurological disorder or medical condition that would compromise subject safety or scan data; treatment within the previous 3 months with medications that might affect the GABA system; use of tobacco products (known to affect GABA levels); alcohol consumption >4 drinks/day; and contraindication to magnetic resonance evaluation.

The following instruments were used for screening: the Structured Clinical Interview for Diagnostic and Statistical Manual of Mental Disorders IV to identify Axis I Disorders and the Time Line Follow Back to assess alcohol consumption. Two (2) reliable and valid psychologic scales were selected to monitor the effects of the interventions on mood and anxiety over time. Mood was assessed with the Exercise- Induced Feeling Inventory (EIFI), which has four subscales: Positive Engagement, Revitalization, Tranquility, and Physical Exhaustion.16 Anxiety was assessed with the State scale of the Spielberger State–Trait Anxiety Inventory (STAI). The EIFI and STAI-State were given before each scan, prior to the first intervention session (week 0), and after completion of sessions at weeks 4, 8, and 12. Metabolic equivalents (METs) are used to rate and compare the physical demands of various activities.18 The American College of Sports Medicine list of metabolic equivalents was consulted to match the 60-minute Iyengar yoga intervention with a 60-minute walking intervention at 2.5 miles per hour (mph) on a flat surface rated at 3.0 METs. During the intervention, the Physical Activity Recall (PAR), a valid and widely used instrument, was used to convert each subject’s weekly physical activity outside of the intervention into a METs score.19,20 For each group, the mean weekly PAR METs scores was computed.

Objectives: Yoga and exercise have beneficial effects on mood and anxiety. g-Aminobutyric acid (GABA)-ergic activity is reduced in mood and anxiety disorders. The practice of yoga postures is associated with increased brain GABA levels. This study addresses the question of whether changes in mood, anxiety, and GABA levels are specific to yoga or related to physical activity.

Methods: Healthy subjects with no significant medical/psychiatric disorders were randomized to yoga or a metabolically matched walking intervention for 60 minutes 3 times a week for 12 weeks. Mood and anxiety scales were taken at weeks 0, 4, 8, 12, and before each magnetic resonance spectroscopy scan. Scan 1 was at baseline. Scan 2, obtained after the 12-week intervention, was followed by a 60-minute yoga or walking intervention, which was immediately followed by Scan 3.

Results: The yoga subjects (nј19) reported greater improvement in mood and greater decreases in anxiety than the walking group (nј15). There were positive correlations between improved mood and decreased anxiety and thalamic GABA levels. The yoga group had positive correlations between changes in mood scales and changes in GABA levels.

Conclusions: The 12-week yoga intervention was associated with greater improvements in mood and anxiety than a metabolically matched walking exercise. This is the first study to demonstrate that increased thalamic GABA levels are associated with improved mood and decreased anxiety. It is also the first time that a behavioral intervention (i.e., yoga postures) has been associated with a positive correlation between acute increases in thalamic GABA levels and improvements in mood and anxiety scales. Given that pharmacologic agents that increase the activity of the GABA system are prescribed to improve mood and decrease anxiety, the reported correlations are in the expected direction. The possible role of GABA in mediating the beneficial effects of yoga on mood and anxiety warrants further study.
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Introduction
Yoga has been used to reduce symptoms of depression, anxiety, and epilepsy. Reduced activity ing-aminobutyric acid (GABA) systems has been found in mood disorders, anxiety disorders, and epilepsy. All three ofthese conditions respond to pharmacologic agents known to increase GABA system activity, raising the possibility that some of the therapeutic effect may be via increased GABA activity. In a previous study using magnetic resonance spectroscopy (MRS) to obtain brain GABA levels, our group demonstrated that experienced yoga practitioners had a significant (27%) increase in whole-slab GABA levels after a 60- minute session of yoga postures compared to no change in GABA levels in controls after a 60-minute reading session. These findings raise the question of whether the associated increase in GABA levels was specific to yoga or related to physical activity in general.

At 84 hours after transfection, apoptotic morphological changes, such as cell rounding, cytoplasmic blebbing, and irregularities of shape, were observed in NB-39-nu cells treated with ALK-siRNAs, whereas no significant changes were seen in the mock-transfected cells or in the luc-siRNA and the s-siRNA treated cells.

These morphological changes were not observed in SK-N-MC cells treated with ALK-siRNAs (data not shown). At 90 hours after transfection, NB-39-nu cells treated with ALK-siRNAs started to detach from the dish due to cell death.
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To examine the localization of expression of ALK kinase, we performed double staining by anti-ALK antibody and TOTO-3, which stains the nucleus, in several neuroblastic cell lines. ALK protein overexpressed in NB-39-nu cells is localized in both membrane and cytoplasm. ALK staining was very weak in cell lines such as YT-nu and SK-N-MC with one copy of the ALK gene, however, its localization appeared to be the same as in NB-39-nu (data not shown). It was observed that the expression of ALK was completely lost after the RNAi-induced suppression of ALK. To confirm whether the cell death resulted from apoptosis, cells were also analyzed by immunofluorescent TUNEL staining in NB-39-nu cells. TUNEL staining was clearly positive in these cells at 84 hours after transfection, indicating that apoptosis was induced in NB-39-nu cells treated with ALK-siRNAs. No significant TUNEL staining was observed in the mock-transfected cells or the luc-siRNA treated cells. Finally, DNA fragmentation assay was performed to measure the endonuclease activity accompanied by apoptosis. The formation of significant DNA fragmentation was observed in the NB-39-nucells but not in SK-N-MC cells treated with ALK-siRNAs, indicating that cell apoptosis was induced through the suppression of ALK only in the NB-39-nu cells. This suggests that signaling pathways downstream of activated ALK dominantly regulate the survival of neuroblastoma cells with amplified ALK; therefore, the loss of ALK protein results in apoptotic changes to these cells.

Expression of ALK in Primary Neuroblastoma Tissues
Immunohistochemically, ALK was positively detected both in the cytoplasm of the neuroblastic cells and in the fine meshwork of neuropil of seven of nine tumors with favorable histology cases with nonamplified N-myc (FH&NA). All seven unfavorable histology tumors (two UH&A tumors and five UH&NA tumors) were positive in the cytoplasm and/or in the fine meshwork of neuropil for ALK. There was no correlation between the frequency or intensity of ALK-staining and histology of neuroblastoma tissues, showing majority of neuroblastoma samples showed a detectable amount of ALK. There was no significant staining using preimmune serum from the same rabbit as that for anti-ALK antibody (data not shown). Essentially the same results were obtained using a mouse monoclonal antibody against human ALK (ALK1: DAKO) (data not shown).

To investigate the effect of suppressing the ALK expression level in ALK-amplified neuroblastoma cells using the RNAi technique, we synthesized two different RNA duplexes directed against nucleotide positions 153 to 171 and 399 to 417 within coding region ALK cDNA (ALK-siRNA1 and ALK-siRNA2, respectively). Because co-transfection of ALK-siRNA1 and ALK-siRNA2 was very effective in suppressing ALK expression, we performed all experiments presented here using a combination of two siRNAs, although similar results were obtained using only ALK-siRNA2. A sequence against the firefly luciferase gene (luc-siRNA) was used as a negative control. The expression of ALK protein is remarkably elevated in NB-39-nu and Nagai compared with other neuroblastoma cell lines, such as SK-M-MC, caused by gene amplification. The RNA duplexes were transfected into NB-39-nu cells with ALK gene amplification and SK-N-MC cells containing only a single copy of the ALK gene. We also tried to introduce ALK-siRNAs in several different neuroblastoma cell lines with or without ALK amplification in addition to NB-39-nu and SK-N-MC cells, resulting in partial or no reduction of ALK expression presumably due to the unsuccessful introduction in those cells. Therefore, we decided to use these two cell lines to perform further analysis of the effect of ALK knockdown by RNAi technique. RT-PCR analysis revealed that ALK mRNA level was reduced in both NB-39-nu cells and SK-N-MC cells treated with ALK-siRNAs, not in the cells treated with luc-siRNA and s-siRNA. Both expression and phosphorylation of ALK kinase were significantly suppressed in the NB-39-nu cells treated with ALK-siRNAs compared with a mock-transfection control or cells treated with luc-siRNA (Figure 2C). In these cells, phosphorylation of ShcC was also suppressed despite the unchanged total amount of ShcC, demonstrating that ShcC is a potent substrate of activated ALK kinase and that activation of ALK is actually responsible for the hyperphosphorylation of ShcC in these cancer cells. While the expression of downstream molecules, such as p44/42 MAPKs and Akt, was not affected by ALK-siRNAs, phosphorylation of these molecules was markedly reduced.

These results suggest that the Ras-MAPK pathway and the phosphatidylinositol 3-kinase/Akt pathway are dominantly regulated by activated ALK kinase in these cells. Interestingly, in SK-N-MC cells treated with ALK-siRNAs, phosphorylation levels of ShcC, p44/42 MAPKs, and Akt were not affected by ALK-siRNAs despite further suppression of the basal ALK expression level, indicating that these pathways are not under the control of ALK in SK-N-MC cells.

As for positive control, tumor xenograft was made by injection of NB-39-nu cells subcutaneously in 5-week-old SCID mice. Immunohistochemical staining with ALK antibody (αALK) (1:1000), was performed on 16 human neuroblastoma tumors selected from the surgical pathology file at the Department of Pathology, Aichi Medical University based on the results of histopathology evaluation31 and N-myc status. All of those tumor samples were obtained before chemotherapy and irradiation therapy and included nine favorable histology cases with nonamplified N-myc (FH&NA), two unfavorable histology cases with amplified N-myc (UH&A), and five unfavorable histology cases with nonamplified N-myc (UH&NA).

Four-micrometer-thick sections from the formalin-fixed and paraffin-embedded tissue samples were deparaffinized and microwaved for three times for 5 minutes in Na-citrate buffer (pH 6.0) for antigen retrieval. The slides were first immersed in 0.3% hydrogen peroxide in methanol for 20 minutes and then in 10% normal goat serum for 30 minutes. The primary antibody (αALK) was then applied at 4°C overnight, followed by a standard staining procedure using the Vectastain ABC kit (Vector Laboratories, Burlingame, CA). Sections were counterstained with hematoxylin for light microscopic review and evaluation. ALK was always positively detected in the cytoplasm of NB-39-nu tumor xenograft and in the cytoplasm and neuritic processes of normal ganglion cells in the separate positive control sections as well as in the test sections as built-in control, whenever available. As for the negative controls, normal rabbit immunoglobulins (1:500 dilution; Vector Laboratories) or preimmune serum for α ALK (1:1000 dilution) was applied as the primary antibody.

Significant Amplification of the ALK Gene and Constitutive Activation of ALK Kinase in Three Neuroblastoma Cell Lines
As shown in Figure 1A, NB-39-nu, Nagai, and NB-1 cells have significant levels of amplification of the ALK gene (30–40 copies per cell) among 25 neuroblastoma and neuroepithelioma cell lines examined. Other cell lines such as SK-N-MC have only one copy of the ALK gene just like the other types of solid tumor cell lines used as controls. In vitro kinase assay revealed outstanding ALK kinase activity in these three cell lines compared with other cells, which is consistent with our previous study. To examine whether overexpressed and activated ALK affects the expression of other RTKs in these cells, protein expression levels of RTKs, including EGFR, Ret, and TrkA, are compared with other cell lines. Significantly high levels of expression of EGFR and TrkA were observed in two of three cell lines overexpressing ALK. Ret expression was commonly elevated in all three cell lines with activated ALK, especially in Nagai and NB-39-nu, consistent with previous study by Northern blotting. Although it is unknown whether overexpression of these RTKs is related to overexpression of ALK, no obvious down-regulation of other RTKs was found in these ALK-amplified cell lines.

For double staining, we first carried out TUNEL and then proceeded to standard immunocytochemistry using anti-ALK antibody. TUNEL was performed using the DeadEnd Fluorometric TUNEL System (Promega) with the following modifications. The NB-39-nu cells seeded on the 24-well plates that were treated with siRNAs were washed with PBS twice and fixed with 4% paraformaldehyde (methanol free) for 25 minutes at 4°C. The cells were rinsed with PBS twice and then permeabilized with 0.2% Triton X-100 solution in PBS for 5 minutes at room temperature. The cells were washed with PBS twice and covered with an equilibration buffer (from the kit) for 10 minutes at room temperature. The equilibration buffer was drained off, and a reaction buffer containing the equilibration buffer, nucleotide mix, and terminal deoxynucleotidyl transferase enzyme was added to the cells and incubated at 37°C for 1 hour, avoiding exposure to light. The cells were incubated for 15 minutes at room temperature with 2× standard saline citrate to stop the reaction. The cells were washed with PBS three times and then stained for ALK using immunofluorescence as follows. The cells were blocked with 2% bovine serum albumin (Boehringer Mannheim) for 30 minutes at room temperature. The blocking solution was drained off, and the cells were incubated with a 1:1000 dilution of αALK for 1 hour at room temperature. The cells were rinsed with PBS three times and incubated with a 1:40 dilution of rhodamine-conjugated goat anti-rabbit secondary antibody (Santa Cruz Biotechnology) for 30 minutes at room temperature. The cells were washed three times with PBS and mounted in glycerol-based 2.5% 1,4-diazabicyclo[2,2,2] octan. Confocal laser scanning analysis was carried out.

DNA Fragmentation Assay
To detect apoptotic DNA cleavage, DNA fragmentation assay was performed using an Apoptotic DNA Ladder kit (Chemicon International, Inc., Temecula, CA). The cells seeded on the 12-well plates that were treated with siRNAs as previously mentioned were collected in 1.5-ml microcentrifuge tubes. The cells were washed with PBS, centrifuged, and lysed with 20 μl of TE lysis buffer. The lysates were incubated with 5 μl of enzyme A (RNase A) at 37°C for 10 minutes and then at 55°C for 30 minutes after the addition of 5 μl of Enzyme B (Proteinase K). Afterward, 5 μl of ammonium acetate solution and 100 μl of absolute ethanol were added, and the samples were kept at −20°C for 10 minutes. The samples were centrifuged, and the pellets were washed with 70% ethanol. Then the DNA pellets were dissolved in 30 μl of DNA suspension buffer. DNA fragmentations were visualized by electrophoresis on 2% agarose gel containing ethidium bromide.

Genomic DNAs derived from neuroblastoma cell lines were obtained from cultured cells as described using the procedure of Perucho et al.29 Samples of 85 neuroblastoma tissues were collected at the Chiba Cancer Center and stored as forms of genomic DNA. The stage criterion was based on the International Neuroblastoma Staging System. Samples of 5 μg of DNA digested by EcoRI were electrophoresed in 0.8% agarose gel and blotted onto nitrocellulose filters (Hybond-N+; Amersham, Piscataway, NJ). The probes for detecting the ALK gene, N-myc gene, and ShcC gene were used in our previous study. The intensities of these signals were measured using a Molecular Imager FxPro (Bio-Rad). This study was approved by the ethical judging committee of the National Cancer Center and the Chiba Cancer Center of Japan.

RNA Interference Technique
Twenty-one-nucleotide double-stranded RNAs were synthesized and purified using Dharmacon Research (Lafayette, CO). To suppress the expression of ALK protein, two different pairs of ALK siRNAs, ALK-siRNA1 and ALK-siRNA2, were obtained. The sequences were 5′-GAGUCUGGCAGUUGACUUCdTdT-3′ for ALK-siRNA1 and 5′-GCUCCGGCGUGCCAAGCAGdTdT-3′ for ALK-siRNA2, corresponding to coding region 153 to 171 and 399 to 417 relative to the first nucleotide of the start codon, respectively. Entire sequences were derived from the sequence of human ALK mRNA (accession no. HSU62540). An siRNA, targeting a sequence in the firefly (Photinus pyralis) luciferase mRNA, was used as a negative control (Dharmacon) (luc-siRNA). We also used a scramble siRNA, Scramble Duplex II (Dharmacon) (s-siRNA) as a mismatch siRNA control in addition to luc-siRNA.

NB-39-nu cells were trypsinized, diluted with growth medium containing 10% fetal calf serum, and transferred to 12-well plates at 6 × 104 cells per well for 24 hours before transfection. The transfection of siRNA was carried out using jetSI (Poly plus transfection). A total of 100 μl of serum-free growth medium and 4 μl of jetSI per well were preincubated for 5 to 10 minutes at room temperature. While the incubation was being performed, 100 μl of serum-free growth medium was mixed with 5 μl of 20 μmol/L siRNA duplex (100 pmol). Total siRNA amounts of 50, 100, and 200 pmol were checked in preliminary experiments to find out 100 pmol is the minimal and optimal amount in this scale of RNAi. The 100 μl of jetSI serum-free medium solution was added to the 100 μl of siRNA duplex solution, gently mixed, and incubated for 30 minutes at room temperature. The growth medium on the cells was removed, and 800 μl of serum-free medium was added to each well. A total of 200 μl of the entire mixture was overlaid onto the cells, and cells were incubated for 4 hours at 37°C in a 5% CO2 incubator. After incubation, 1 ml of medium containing 4% fetal calf serum was added without removing the transfection mixture (final concentration 2%). The cells were assayed 84 hours after transfection. SK-N-MC cells were seeded in 12-well plates at a concentration of 1.3 × 105 cells per well. These were treated with siRNAs in the same way as NB-39-nu and assayed 48 hours after transfection. In the 24-well plate, the cells were seeded at the same concentration as the 12-well plate, and siRNAs and all other reagents were used at half volume. After transfection, the cells were examined under a light microscope every day.

Immunoprecipitation and immunoblotting were performed as described previously. The polyclonal antibodies against the CH1 domains of ShcC (amino acids 306–371) and the anti-ALK antibody (αALK) that was against the cytoplasmic portion (amino acid 1379–1524) of human ALK were prepared as described previously. An anti-phosphotyrosine antibody (4G10) was obtained from UBI. Anti-p44/42 MAPKs, anti-phospho-p44/42 MAPKs, anti-Akt, and anti-phospho-Akt antibodies were purchased from Cell Signaling (Beverly, MA). Anti-EGF receptor (EGFR), anti-Ret, and anti-TrkA antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). In vitro kinase assay for ALK was performed as previously described. Anti-ALK immunoprecipitates were incubated with or without Poly-Glu/Tyr as an exogenous substrate.

Immunocytostaining
For ALK/TOTO-3, immunostaining using anti-ALK antibody was performed at first, and then nuclei were stained using TOTO-3. The cells seeded on the 24-well plates were washed with phosphate-buffered saline (PBS) three times and fixed with 4% paraformaldehyde (methanol free) for 5 minutes at room temperature. The cells were rinsed with PBS twice and then permeabilized with 0.2% Triton X-100 solution in PBS for 10 minutes at room temperature. The cells were blocked with 5% goat serum and 3% bovine serum albumin–Tris-buffered saline for 30 minutes at room temperature. The blocking solution was drained off, and the cells were incubated with a 1:1000 dilution of αALK for 1 hour at room temperature. The cells were rinsed with PBS three times and incubated with a 1:2000 dilution of Alexa fluor (Molecular Probes, Eugene, OR) and 1: 100 dilution of TOTO-3 (Molecular Probes) for 30 minutes at room temperature. The cells were washed three times with PBS and mounted in glycerol-based 2.5% 1,4-diazabicyclo[2,2,2] octan. Confocal laser scanning analysis was carried out. For ALK/TUNEL, we first carried out TUNEL and then proceeded to standard immunocytochemistry using anti-ALK antibody. TUNEL was performed using the DeadEnd Fluorometric TUNEL System (Promega, Madison, WI) with the following modifications. The NB-39-nu cells seeded on the 24-well plates that were treated with siRNAs were washed with PBS twice and fixed with 4% paraformaldehyde (methanol free) for 25 minutes at 4°C. The cells were rinsed with PBS twice and then permeabilized with 0.2% Triton X-100 solution in PBS for 5 minutes at room temperature. The cells were washed with PBS twice and covered with an equilibration buffer (from the kit) for 10 minutes at room temperature. The equilibration buffer was drained off, and a reaction buffer containing the equilibration buffer, nucleotide mix, and terminal deoxynucleotidyl transferase enzyme was added to the cells and incubated at 37°C for 1 hour, avoiding exposure to light. The cells were incubated for 15 minutes at room temperature with 2× standard saline citrate to stop the reaction. The cells were washed with PBS three times and then stained for ALK using immunofluorescence as follows. The cells were blocked with 2% bovine serum albumin (Boehringer Mannheim, Germany) for 30 minutes at room temperature. The blocking solution was drained off, and the cells were incubated with a 1:1000 dilution of αALK for 1 hour at room temperature. The cells were rinsed with PBS three times and incubated with a 1:40 dilution of rhodamine-conjugated goat anti-rabbit secondary antibody (Santa Cruz Biotechnology) for 30 minutes at room temperature. The cells were washed three times with PBS and then mounted and observed in the same manner as that for ALK/TOTO-3.

Pathologists characterize many aspects of breast cancer specimens but rarely pay much attention to the stroma. Whereas some tumors have more stroma than others, desmoplasia is only one of many properties that vary among individual ductal carcinomas. It seems logical that the pathology assessment should identify risk factors associated with rapid tumor progression; however, the pathologist focuses on issues that are more pragmatic: the extent of tumor, classification into histologic subtypes, and identification of properties that define therapeutic intervention, such as expression of estrogen receptor (ER) or HER2/neu. In this issue of The American Journal of Pathology, Conklin et al1 have demonstrated a stromal signature that has a correlation with progression. Although the potential exists for this signature to be used for prognosis, the main value of their correlation is to identify two important areas for further investigation. First, the stromal signature indicates a biologically significant process because it predicts how the tumor will behave. Defining this process is the first step in learning how to control it. Second, the stromal signature may indicate a novel mechanism of tumor progression if it is independent of other prognostic markers.

Multiphoton Microscopy

The new study is based on research from the Keely laboratory and others who used multiphoton microscopy to examine the structure of collagen fibers in tumors. Multiphoton microscopy (or two-photon microscopy) utilizes lasers that generate pulses of light with extremely high photon density in the near-infrared wavelength range of 700 to 1300 nm. For fluorescence imaging, the photon density is so high that near-simultaneous absorption of two photons by a fluorophore can occur, resulting in activation that is equivalent to absorption of a single photon with twice the energy. For example, with the laser tuned to 920 nm, simultaneous absorption of two photons is equivalent to absorption of a single 460-nm photon that can thus activate green fluorescent protein, which has a broad absorption peak around 500 nm. The nonlinear dependence of this effect on the square of photon density makes multiphoton fluorescence microscopy confocal in nature. It is also more efficient than ordinary fluorescence microscopy; there is less bleaching of fluorophores out of the plane of focus and deeper penetration into tissues because of the reduced scattering of near-infrared photons.

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To image unstained collagen fibers in the tumor, Conklin et al used second harmonic generation, an additional but less well known advantage of multiphoton imaging. In second harmonic generation, periodic structures that are not centrosymmetriceg, collagen fibers—can act like frequency doubling crystals. In essence, two photons of one frequency enter the fiber, and one photon with exactly half the frequency leaves it. These second harmonic signals are mostly emitted in the same direction as the excitation light, but some are scattered backward and detected by the microscope used to deliver the excitation signal. The second harmonic signal is dependent on the presence of appropriately polarized structures including collagen fibers, myosin, and even microtubules.

The second harmonic signal for collagen fibers is detectable in paraffin sections of formalin-fixed tissue. Thus Conklin et al examined a standard breast cancer tissue microarray. The orientation of the collagen fibers was determined relative to the tumor cell masses to generate what the authors term the “tumor-associated collagen signature” or TACS. Different TACS categories correspond to various ways that the extracellular matrix can be organized with respect to the tumor cells. Originally these categories were defined by studying mammary tumor development in mouse models. TACS-1 arises first in early tumors with increased numbers of curved, apparently relaxed collagen fibers around the tumor. TACS-2 develops as the tumor grows larger. The surrounding fibers become straight and parallel to the surface of the tumor, probably reflecting stretching of the fibers due to the expansion of the tumor. TACS-3 reflects a significant reorganization of the matrix, so that straight matrix fibers now lead directly into the tumor cell mass. In TACS-3, the fibers can act as pathways along which cells can crawl, as has been seen in multiphoton imaging of metastatic tumors.