Cyclic phosphatidic acid inhibits RhoA-mediated autophosphorylation of FAK at Tyr-397 and subsequent tumor-cell invasion

We demonstrated previously that rat ascites hepatoma MM1 cells require both lysophosphatidic acid (LPA) and fibronectin (FN) for phagokinetic motility and transcellular migration and that these events are regulated through the RhoA-ROCK pathway and tyrosine phosphorylation of proteins including focal adhesion kinase (FAK). Moreover, we reported that palmitoyl-cyclic phosphatidic acid (Pal-cPA), a structural analogue of LPA, inhibits LPA-induced migration of MM1 cells and experimental metastasis of B16 murine melanoma cells. However, the molecular mechanisms of action of Pal-cPA remains to be clarified. To examine this, total cellular lysates after stimulation with LPA or FN were subjected to time-course immunoblot analysis with anti-phophotyrosine and anti-pY397-FAK antibodies. Tyrosine-phosphorylation of FAK especially at Tyr-397 was obviously persistent after stimulation with LPA + FN compared to after stimulation with LPA alone. This persistent phosphorylation was necessary for MM1 cell migration and inhibited by Pal-cPA as by C3 exoenzyme Rho inhibitor. RhoA activity (GTP-bound RhoA) was also measured by the pull down assay using the Rho binding domain of Rhotekin. LPA-induced RhoA-activation of MM1 cells was completely inhibited by Pal-cPA. Moreover, we demonstrated that autophosphorylation of FAK at Tyr-397, downstream of RhoA, contributed to formation of focal adhesions and was critical in LPA-induced MM1 cell migration by developing autophosphorylation-deficient (Y397F) FAK-transfectants. Collectively, Pal-cPA hampered LPA-induced morphological changes and transcellular migration of MM1 cells through downregulating active RhoA and inhibiting its downstream events including autophosphorylation of FAK. Pal-cPA also inhibited endogenous (LPA-independent) activation of RhoA in human fibrosarcoma HT-1080 cells. Pal-cPA may potentially provide a new therapy for the treatment of cancer invasion and metastasis.     

Accurate pathological staging of urothelial neoplasms requires better cystoscopic sampling

PURPOSE: The frequency with which muscularis propria was sampled by urologists and the sources of interpretive discrepancies among pathologists were studied in a community practice setting. MATERIALS AND METHODS: A total of 217 consecutive cases of urothelial neoplasm were independently reviewed by 3 pathologists. The presence or absence of muscularis propria as well as interpretive discrepancies were recorded. RESULTS: Despite clinical emphasis on accurate pathological staging portions of muscularis propria were absent from samples of histologically documented urothelial neoplasms in up to 51% of cases. Failure to obtain muscularis propria varied widely among urologists but was most often associated with cases of low grade papillary neoplasms, in which invasion is less likely. Muscularis propria was usually present in cases of noninvasive carcinoma in situ but this may have represented inadvertent sampling of structures in close proximity. The incidence of interpretive discrepancies among pathologists who are required to assess the status of muscularis propria was significant (24%). Almost all problems were related to artifacts and most may have been avoided if careful attention had been given to specimen sampling and processing. CONCLUSIONS: The well documented tendency toward cystoscopic under staging has not necessarily resulted in a high incidence of muscularis propria in bladder cases of urothelial neoplasms. Even when muscle may have been sampled, artifacts that were often due to thermocoagulation hampered accurate pathological staging.     

Failure to find evidence for association between voltage-gated sodium channel gene SCN2A variants and febrile seizures in humans

The voltage-gated sodium channel type II alpha polypeptide gene (SCN2A) R188W mutation with channel dysfunction was recently identified in a patient with febrile and afebrile seizures. A possible association between SCN2A R19K polymorphism and febrile seizures (FS) associated with afebrile seizures including generalized epilepsy with febrile seizures plus (GEFS+) was also noted. We attempted to identify the R188W mutation and confirm association of the R19K polymorphism in 93 Japanese patients with FS, 35 Japanese patients with FS associated with afebrile seizures including GEFS+, and 100 control subjects. The R188W mutation was not found. There were no significant differences in genotype or allele frequencies of the R19K polymorphism between groups. Our study failed to provide evidence supporting a causal relation between the SCN2A mutation/polymorphism and FS or FS associated with afebrile seizures including GEFS+ in the Japanese population.     

Glucocorticoid receptors in normal human eosinophils: comparison with neutrophils

Saturation analysis with [³H]-dexamethasone was employed to measure glucocorticoid binding in purified preparations of human eosinophils and neutrophils. Eosinophils contained 10.8 ± 1.3 × 10³ high-affinity receptor sites per cell, with a dissociation constant (K_d) of 15.3 ± 0.6 nM dexamethasone. Cortisol was capable of competing with [³H]-dexamethasone in the binding reaction, whereas progesterone, estradiol, estriol, and testosterone were less effective. Saturable glucocorticoid binding in neutrophils had a K_d of 17.7 ± 0.8 nM dexamethasone with 11.1 ± 0.8 × 10³ sites per cell and displayed similar steroid specificity. These data indicate that normal human eosinophils have glucocorticoid receptors with characteristics similar to those in neutrophils and that in these cells ligand-receptor interaction can occur at physiologic glucocorticoid concentrations. Furthermore, these results suggest that certain glucocorticoid effects on eosinophils and neutrophils may be mediated through specific receptors.     

Nanofabrication for micropatterned cell arrays by combining electron beam-irradiated polymer grafting and localized laser ablation

Most methods reported for cell-surface patterning are generally based on photolithography and use of silicon or glass substrates with processing analogous to semiconductor manufacturing. Herein, we report a novel method to prepare patterned plastic surfaces to achieve cell arrays by combining homogeneous polymer grafting by electron beam irradiation and localized laser ablation of the grafted polymer. Poly(N-isopropylacrylamide) (PIPAAm) was covalently grafted to surfaces of tissue culture-grade polystyrene dishes. Subsequent ultraviolet ArF excimer laser exposure to limited square areas (sides of 30 or 50 microm) produced patterned ablative photodecomposition of only the surface region (approximately 100-nm depth). Three-dimensional surface profiles showed that these ablated surfaces were as smooth and flat as the original tissue culture-grade polystyrene surfaces. Time-of-flight secondary ion mass spectrometry analysis revealed that the ablated domains exposed basal polystyrene and were surrounded with PIPAAm-grafted chemistry. Before cell seeding, fibronectin was adsorbed selectively onto ablated domains at 20 degrees C, a condition in which the non-ablated grafted PIPAAm matrix remains highly hydrated. Hepatocytes seeded specifically adhered onto the ablated domains adsorbed with fibronectin. Because PIPAAm, inhibits cell adhesion and migration even at 37 degrees C when the grafted density is > 3 microg/cm2, all the cells were confined within the ablated domains. A 100-cell domain array was achieved by this method. This surface modification technique can be utilized for fabrication of cell-based biosensors as well as tissue-engineered constructs.     

Differentiation from embryonic stem cells to vascular wall cells under in vitro pulsatile flow loading

This study evaluated the possibility of differentiation from embryonic stem (ES) cells to vascular wall cells by physical (mechanical) stress loading in vitro. A cell mixture containing Flk1-positive cells (ca. 30%) derived from murine ES cells was added to a compliant microporous tube made of segmented polyurethane. The compliance of the tube was close to that of the human artery [the stiffness parameter (β) = 57.2 (n = 5, SD < 5%)]. The luminal surface of the tube was fully covered with the cells by preincubation for two days in the presence of vascular endothelial growth factor (VEGF). After 2 days of additional incubation without VEGF under static conditions, layering of the grown cells, mostly smooth muscle actin (SMA)-positive cells, was observed only on the luminal surface of the tube. The cells were flat, polygonal, and randomly oriented. On the other hand, after a 2-day incubation under a weak pulsatile flow simulating the human venous systems [wall shear stress (WSS) from −0.98 to 2.2 dyn/cm²; circumferential strain (CS) 4.6–9.6 × 10⁴ dyn/cm²] without VEGF, cells in the superficial layer were regularly oriented in the direction of the pulsatial flow. The oriented cells exhibited endothelial-like appearance, indicating that they were platelet endothelial cell adhesion molecule 1 (PECAM1)-positive. In addition, the cells growing into the interstices in the deeper layer showed smooth muscle-like appearance, indicating that they were SMA-positive. Differentiation to two different cell types and segregation of incorporated ES cells may be simultaneously encouraged by the combination of WSS and CS. It is expected that the monobloc building of hierarchically structured hybrid vascular prostheses composed of several vascular wall cell types is possible by physically synchronized differentiation of ES cells.     

Adrenal Schwannoma in an Elderly Man: A Case Report and Literature Review

Schwannoma is a common mesenchymal neoplasm; however, adrenal schwannoma is rare, and it is frequently misdiagnosed as adrenal cortical adenoma. We herein report a 91-year-old Japanese man with right adrenal schwannoma that was pathologically diagnosed after adrenalectomy. To our knowledge, this is the first case of adrenal schwannoma in the oldest patient and with the longest follow-up period reported, including radiological images from 10 years earlier.     

Comparative efficacies of different antibiotic treatments to eradicate nontypeable <Emphasis Type="Italic">Haemophilus influenzae</Emphasis>infection

Background  

Nonencapsulated and nontypeable Haemophilus influenzae (NTHi) is a major cause of human respiratory tract infections. Some strains of NTHi can cause invasive diseases such as septicemia and meningitis, even if H. influenzae is not generally considered to be an intracellular pathogen. There have been very few reports about the therapeutic efficacy of antibiotics against respiratory tract infection caused by NTHi in mice because it is difficult for H. influenzae to infect mice. Therefore, we evaluated the efficacy of antibiotics against NTHi in both a cell culture model and a mouse model of infection.