Frequent co-localization of Cox-2 and laminin-5 gamma2 chain at the invasive front of early-stage lung adenocarcinomas

Laminin-5 is an extracellular matrix protein that plays a key role in cell migration and tumor invasion. Cox-2 is an induced isoform of cyclooxygenases that plays an important role in carcinogenesis, suppression of apoptosis, angiogenesis, and metastasis of colon cancer. We report frequent co-expression of cox-2 and laminin-5 at the invasive front of early-stage lung adenocarcinomas. We investigated the expression of cox-2 and laminin-5 immunohistochemically in 102 cases of small-sized lung adenocarcinoma (maximum dimension, 2 cm or less). Cox-2 and laminin-5 were expressed in 97 (95.1%) and 82 (80.4%) cases, respectively. Both were preferentially localized in cancer cells at the cancer-stroma interface, although cox-2 tended to show a diffuse staining pattern in some cases. A comparison of their staining patterns revealed a striking similarity in their distribution in 24 cases, and a partial overlap between their localization in another 20 cases. Moreover, an overall correlation was found between the expression levels of cox-2 and laminin-5 (P = 0.018). To gain insight into the mechanisms that regulate the expression of these proteins, we additionally studied their expression in 58 cases of stage I lung adenocarcinoma, in which p53 status was determined by immunohistochemistry, polymerase chain reaction-single strand conformation polymorphism analysis, and direct sequencing. The results showed that tumors with mutant p53 tended to express more cox-2 than those with wild-type p53 (P = 0.080). Also, tumors that overexpressed p53 had higher levels of cox-2 and laminin-5 than those without p53 overexpression (P = 0.032 and 0.047, respectively). Further immunohistochemical analysis showed that tumors that overexpressed both epidermal growth factor receptor (EGFR) and erbB-2 had higher levels of cox-2 and laminin-5 than those without concomitant overexpression of these proteins (P = 0.014 and P = 0.018, respectively). To see whether EGFR signaling is involved in cox-2 and laminin-5 expression, we further conducted in vitro analyses using six lung adenocarcinoma cell lines (A549, HLC-1, ABC-1, LC-2/ad, VMRC-LCD, and L27). Western blot analyses showed that cox-2 mRNA levels, and to a lesser extent laminin-5 gamma2 mRNA levels, correlated with the expression levels of erbB-2 and the phosphorylated form of MAPK/ERK-1/2 protein. The addition of transforming growth factor-alpha increased both cox-2 and laminin-5 gamma2 mRNA levels in A549, ABC-1, and L27 with different kinetics; the induction of cox-2 occurred earlier than that of laminin-5 gamma2. Finally, the migration of ABC-1 cells was inhibited by MAP kinase kinase inhibitor PD98059 and a selective cox-2 inhibitor NS-398. In contrast, the migration of A549 cells was inhibited by PD98059, but much less effectively by NS-398. These results suggest that co-stimulatory mechanisms may exist that increase the expression of cox-2 and laminin-5 at the invasive front of lung adenocarcinomas and that EGFR signaling could be one of the mechanisms. Further investigations are warranted concerning the role of cox-2 and laminin-5 in cancer cell invasion and the significance of p53 and EGFR signaling in the regulation of cox-2 and laminin-5 expression.     

Specific tension of elbow flexor and extensor muscles based on magnetic resonance imaging

Series cross-section images of the upper extremity were obtained for four men by magnetic resonance imaging (MRI) and anatomical cross-sectional areas (ACSA) of elbow flexor muscles [biceps brachii (BIC), brachialis (BRA), brachioradialis (BRD)] and extensor muscles [triceps brachii (TRI)] were measured. Physiological cross-sectional area (PCSA) was calculated from the muscle volume and muscle fibre length, the former from the series ACSA and the latter from the muscle length multiplied by previously reported fibre/muscle length ratios. Elbow flexion/extension torque was measured using an isokinetic dynamometer and the force at the tendons was calculated from the torque and moment arms of muscles measured by MRI. Maximal ACSA of TRI was comparable to that of total flexors, while PCSA of TRI was greater by 1.9 times. Within flexors, BRA had the greatest contribution to torque (47%), followed by BIC (34%) and BRD (19%). Specific tension related to the estimated velocity of muscle fibres were similar for elbow flexors and extensors, suggesting that the capacity of tension development is analogous between two muscle groups.     

Three-dimensional two-layer collagen matrix gel culture model for evaluating complex biological functions of monocyte-derived dendritic cells

Dendritic cell-like cells (Mo-DCs) generated from peripheral blood monocytes with interleukin-4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) have been used as tools to treat cancer patients (DC-vaccines). Because Mo-DCs have multiple antigen presentation-related functions, including phagocytosis, migration, cytokine production, and T cell stimulation, establishment of a method for simultaneously evaluating the various functions of Mo-DCs is important. We developed a new in vitro three-dimensional two-layer collagen matrix culture model that consists of a collagen gel containing Mo-DCs as the lower layer and a collagen gel containing necrotic GCTM-1 tumor cells and/or T cells as the upper layer. We used this system to observe simultaneously multiple functions of Mo-DCs by phase-contrast or fluorescence microscopy and to assess IL-12 secretion during more than 2 weeks of culture. We also observed interactions between Mo-DCs and necrotic GCTM-1 or T cells on an individual cell basis by time-lapse videomicroscopy. In addition, we collected Mo-DCs from the collagen gels by collagenase treatment and analyzed the expression of antigen presentation-related molecules such as HLA-DR, CD80, CD83, and CD86 on Mo-DCs. This model may be a useful tool for evaluation of the various functions of Mo-DCs used as DC vaccines and for studies of the complex behaviors of Mo-DCs in vivo.     

Helicobacter pylori seropositivity and IL-1B C-31T polymorphism among Japanese Brazilians

We reported previously that anti-Helicobacter pylori antibody seropositivity (HP+) had an association with interleukin 1B (IL-1B) C-31T genotype, especially among smokers. This study examined the association for Japanese Brazilians. In this cross-sectional study, voluntary participation was announced through Japanese Brazilian communities in Sao Paulo, Curitiba, Mogi das Cruzes, and Mirandopolis; 963 Japanese Brazilians (399 males and 564 females) aged 33-69 years participated. Lifestyle data and peripheral blood were collected. An anti-HP IgG antibody test and genotyping for IL-1B C-31T and IL-1RN 86 bp VNTR were independently conducted. The genotype frequency of the IL-1B polymorphism among 947 individuals was 23.9% for C/C genotype, 45.6% for C/T genotype, and 30.5% for T/T genotype. Sex-age-adjusted odds ratio (aOR) of HP+ was 1.30 (95% confidence interval, 0.94-1.81) for C/T genotype and 1.45 (1.02-2.07) for T/T genotype relative to C/C genotype. The aOR for 127 current smokers was 2.45 (0.91-6.55) for C/T and 3.49 (1.17-10.46) for T/T, while that for 667 never smokers was 1.21 (0.82-1.78) and 1.36 (0.90-2.05), respectively. The corresponding figures were 2.42 (1.16-5.02) and 3.00 (1.33-6.78) for 226 current drinkers, and 1.21 (0.82-1.78) and 1.36 (0.90-2.05) for 667 non-drinkers. The difference in the OR was observed for milk consumption, salty pickled vegetable eating, and physical exercise practice. 4/4 Genotype of IL-1RN 86 bp VNTR was 84.8%, and had no association with the HP seropositivity. The observed association between HP+ and IL-1B -31TT indicated that the genetic trait also influences the susceptibility to HP for Japanese in Brazil.     

De novo interstitial deletion of 4q[46,XX,del(4)(q27q28.2)] with intact blood group-MN locus,confining its locus to 4q28.2–4q31.1

We report a malformed female infant withde novo interstitial deletion of 4q[46,XX,del(4)(q27q28.2)]. The MN blood type analysis of the family members showed that the patient had an intact blood group-MN locus. The locus of the gene responsible for the MN antigen activity is confined to a 4q28.2–4q31.1 segment on the basis of the result of this patient and the previous mapping data.     

Cell to cell contact through ICAM-1-LFA-1 and TNF-alpha synergistically contributes to GM-CSF and subsequent cytokine synthesis in DBA/2 mice induced by 1,3-beta-D-Glucan SCG.

SCG is a major 6-branched 1,3-beta-D-glucan in Sparassis crispa Fr. showing antitumor activity. We recently found that the splenocytes from naive DBA/1 and DBA/2 mice are potently induced by SCG to produce interferon- gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin-12p70 (IL-12p70), and that GM-CSF plays a key biologic role among these cytokines. In this study, we investigated the contribution of cell-cell contact and soluble factors to cytokine induction by SCG in DBA/2 mice. Cell-cell contact involving intercellular adhesion molecule-1 (ICAM-1) and lymphocyte function-associated antigen-1 (LFA-1) was an essential step for the induction of GM-CSF and IFN-gamma by SCG but not for the induction of TNF-alpha or IL-12p70 by SCG. SCG directly induced adherent splenocytes to produce TNF-alpha and IL-12p70. GM-CSF was required for the induction of TNF-alpha by SCG, and in turn, TNF-alpha enhanced the release of GM-CSF and thereby augmented the induction of IL-12p70 and IFN-gamma by SCG. Neutralization of IL-12 significantly inhibited the induction of IFN-gamma by SCG. We concluded that induction of GM-CSF production by SCG was mediated through ICAM-1 and LFA-1 interaction, GM-CSF subsequently contributed to further cytokine induction by SCG, and reciprocal actions of the cytokines were essential for enhancement of the overall response to SCG in DBA/2 mice.     

Membrane potential changes in vocal cord tensor motoneurons during breathing, vocalization, coughing and swallowing in decerebrate cats

We studied the patterns of membrane potential changes in vocal cord tensor motoneurons, i.e. cricothyroid muscle motoneurons (CTMs), during fictive breathing, vocalization, coughing, and swallowing in decerebrate paralyzed cats to determine the nature of central drives to CTMs during these behaviors. CTMs were identified by antidromic activation from the superior laryngeal nerve. During breathing, CTMs always depolarized during the inspiratory phase, and sometimes depolarized during the expiratory phase as well. During vocalization, CTMs strongly depolarized. During coughing, CTMs exhibited depolarizations during both inspiratory and expiratory phases, but it was interrupted by a transient repolarization between the last part of the inspiratory phase and the first part of the abdominal burst during which chloride-dependent inhibitory postsynaptic potentials were revealed. During swallowing, most CTMs hyperpolarized, and this hyperpolarization was sometimes followed by a weak depolarization. We conclude that the main role of the cricothyroid muscle is vocalization but the functional roles in coughing and swallowing are minor, and that the CTM activity during resting breathing and vocalization are primarily controlled by excitatory inputs, while during coughing and swallowing, inhibitory inputs play roles in shaping membrane potential trajectories.     

Application of Scanning Acoustic Microscopy for Assessing Stress Distribution in Atherosclerotic Plaque

Scanning acoustic microscopy (SAM) was equipped to assess the acoustic properties of normal and atherosclerotic coronary arteries. The SAM image in the atherosclerotic lesion clearly demonstrated that the sound speed was higher than that in the normal intima, and that the variation of elasticity was found within the fibrous cap of the plaque. Young's elastic modulus of each region was calculated and the finite element analysis was applied to derive the stress distribution in these arterial walls. In a case of normal coronary artery, the stress was dominant in the intima and the distribution was rather homogeneous and in a case of atherosclerosis, high stress was concentrated to the relatively soft lesion in the fibrous cap overlying lipid pool. SAM provides information on the physical properties, which cannot be obtained by the optical microscope. The results would help in understanding the pathological features of atherosclerosis. © 2001 Biomedical Engineering Society. PAC01: 8764-t, 8763Df, 8719Xx, 8719Rr     

Involvement of Cdc42 and Rac small G proteins in invadopodia formation of RPMI7951 cells

BACKGROUND: Invadopodia are membrane protrusions into the extracellular matrix by aggressive tumour cells. These structures are associated with sites of matrix degradation and invasiveness of malignant tumour cells in an in vitro fibronectin degradation/invasion assay. The Rho family small G proteins, consisting of the Rho, Rac and Cdc42 subfamilies, are implicated in various cell functions, such as cell shape change, adhesion, and motility, through reorganization of the actin cytoskeleton. We studied the roles of the Rho family small G proteins in invadopodia formation. RESULTS: We first demonstrated that invadopodia of RPMI7951 human melanoma cells extended into the matrix substratum on a vertical view using a laser scanning confocal microscope system. We confirmed that invadopodia were rich in actin filaments (F-actin) and visualized clearly with F-actin staining on a vertical view as well as on a horizontal view. We then studied the roles of Rho, Rac, and Cdc42 in invasiveness of the same cell line. In the in vitro fibronectin degradation/invasion assay, a dominant active mutant of Cdc42 enhanced dot-like degradation, whereas a dominant active mutant of Rac enhanced diffuse-type degradation. Furthermore, frabin, a GDP/GTP exchange protein for Cdc42 with F-actin-binding activity, enhanced both dot-like and diffuse-type degradation. However, a dominant active mutant of Rho did not affect the fibronectin degradation. Moreover, inhibition of phosphatidylinositol-3 kinase (PI3K) disrupted the Rac and Cdc42-dependent actin structures and blocked the fibronectin degradation. CONCLUSION: These results suggest that Cdc42 and Rac play important roles in fibronectin degradation and invasiveness in a coordinate manner through the frabin-Cdc42/Rac-PI3K signalling pathway.