Elevated Cell Invasion Is Induced by Hypoxia in a Human Pituitary Adenoma Cell Line

Pituitary adenoma tissues are hypovascular, and have a lower partial oxygen pressure compared with neighboring normal organs. In this study, we investigated whether hypoxia influences the cell invasiveness of the human pituitary adenoma cell line, HP-75. HP-75 cells were exposed to hypoxic (1–10% oxygen) or normoxic (21% oxygen) conditions for 24 hours. Gelatin and reverse zymogram assays were used to determine the enzyme activities of matrix metalloproteinases (MMP) and tissue inhibitor of metalloproteinases (TIMP). Cell adhesion and Matrigel cell invasion were examined with a Boiden chamber. Finally, the mRNA gene expression profiles of cells exposed to hypoxia or normoxia were examined by cDNA microarray and confirmed with real-time RT-PCR and flow cytometry. The gelatin and reverse zymograms revealed that the activities of MMP and TIMP were not significantly altered by hypoxia. Matrigel cell invasion and cell adhesion to Matrigel or collagen type IV were increased by hypoxia (3.8- and 4.8-fold, respectively). The cDNA microarray analysis revealed that laminin β2 chain mRNA was specifically up-regulated under hypoxic conditions (4.96-fold). Finally, real-time RT-PCR and flow cytometry verified the elevated expression of laminin β2 chain at the mRNA and protein levels under hypoxic conditions. RNA interference with siRNA targeting laminin β2 inhibited Matrigel invasion and adhesion to collagen type IV in a dose.dependent manner.Collectively, these results suggested that hypoxia (1% oxygen) enhanced the cell invasion properties of a pituitary adenoma cell line in association with elevated expression of laminin β2 and enhanced binding to collagen type IV.Key Words: cell invasion, hypoxia, laminin β2, pituitary adenoma, siRNA     

A simple intact protein analysis by MALDI-MS for characterization of ribosomal proteins of two genome-sequenced lactic acid bacteria and verification of their amino acid sequences

Rapid identification of bacteria by a bioinformatics-based approach, which processes the mass spectra observed by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS), relies on the calculated masses of ribosomal subunit proteins as biomarkers predicted from amino acid sequences found in protein sequence databases. To verify the actual state of the registered sequence information, a simple intact protein analysis by MALDI-MS using cell lysates as samples was applied to the characterization of ribosomal proteins from genome-sequenced Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus strains. This method avoided the risk of loss of some subunit proteins and the formation of disulfide bonds during the purification of ribosomal proteins. By comparing this with the MALDI mass spectra of different strains and carrying out manual inspection of sequence information, a total of five errors in N-terminal amino acid sequences were identified. After sequence correction, approximately 40 out of 53 subunit proteins could be assigned, considering N-terminal methionine loss only as a post-translational modification. These show promise for use as practical biomarkers for the rapid identification of S. thermophilus and L. bulgaricus. After verification of these amino acid sequences, mass differences relative to those of genome-sequenced strains have the potential for distinguishing bacteria at the strain level.     

SH-containing cellulose acetate derivatives: preparation and characterization as a shape memory-recovery material

Cellulose derivatives having a cross-linkable mercapto group were prepared by esterification of cellulose acetate (CA) with mercaptoacetic acid. The molecular structure of a series of products (CA-MA) was characterized by (1)H and (1)H- (13)C HMQC NMR spectroscopy and gel permeation chromatography. The solubility of CA-MA in water and organic solvents could be controlled by changing the preparation conditions including the degree of acetyl substitution of the starting CA. The CA-MA samples thus synthesized showed a sol-gel transition in solution and a shape memory-recovery behavior in film form through adequate redox treatments due to the reversible, cross-linking association and dissociation between mercapto groups. Dimethyl sulfoxide was usable as the organic solvent and oxidant, while the major reducing reagent was 2-mercaptoethanol or ammonium mercaptoacetic acid. The progress of the redox reactions was followed by using a confocal depth scanning technique in Raman spectroscopy. It was found that the compatibility between the cellulose derivatives and the redox reagents used was an important factor for the successful reactions, especially in the samples of film form. The cross-linking effect on the thermal and viscoelastic properties of the CA-MA films was also estimated by differential scanning calorimetry and dynamic mechanical analysis. Discussion focused on the alternately declining and recovering behavior of a principal loss tan delta peak, observed following the redox treatments repeated for the CA-MA film specimens.     

Individual Variation in Behavioural Reactions to Unfamiliar Conspecific Vocalisation and Hormonal Underpinnings in Male Chimpanzees

It has been established that various species exhibit personality, defined as intra‐individual consistency and inter‐individual variation in behavioural phenotypes. For example, certain individuals may demonstrate consistently greater behavioural reactions and elevated stress responses. We conducted playback experiments and hormonal analyses on male chimpanzees (Pan troglodytes) in captivity to investigate the patterns and proximate mediators of individual variations in behavioural reactions. We found intra‐individual consistency and inter‐individual variation in behavioural reactions (intensive vigilance towards the direction of speakers) to vocalisations by unfamiliar chimpanzees. This behavioural reaction was positively correlated with changes in salivary cortisol concentration, suggesting that stress is a proximate factor mediating the variation in behavioural reactions. The males who were highly responsive to the conspecific vocalisation also exhibited high behavioural reactions towards the neutral broadcast stimulus (the jungle crow’s Corvus macrorhynchos ‘ka’ vocalisation). This observation is consistent with the notion that male chimpanzees vary in intrinsic behavioural tendency to different stimuli.     

Enzymatic saccharification of woody biomass micro/nanofibrillated by continuous extrusion process I--effect of additives with cellulose affinity

Mechanical micro/nanofibrillation of Douglas fir was performed by a continuous extrusion process in an attempt to develop a cost-effective pretreatment method for enzymatic saccharification. Additives with cellulose affinity (ethylene glycol, glycerol, and dimethyl sulfoxide) were used to effectively fibrillate the wood cell wall into submicron- or nano-scale, thus opening up the cell wall structure for improving enzymatic accessibility, and lower the extrusion torque. Morphological characterization showed that ethylene glycol was the most effective additive for fibrillation. The fibrillated products were converted into glucose with a high yield by enzymatic saccharification. The maximum cellulose-to-glucose conversion was achieved when ethylene glycol was used; the value was 62.4%. The glucose yield was approximately 6 times higher than that of the untreated raw material.     

RB1CC1 together with RB1 and p53 predicts long-term survival in Japanese breast cancer patients

RB1-inducible coiled-coil 1 (RB1CC1) plays a significant role in the enhancement of the retinoblastoma tumor suppressor (RB1) pathway and is involved in breast cancer development. However, RB1CC1's role in clinical progression of breast cancer has not yet been evaluated, so, as a first step, it is necessary to establish its usefulness as a tool to evaluate breast cancer patients. In this report, we have analyzed the correlation between abnormalities in the RB1CC1 pathway and long-term prognosis, because disease-specific death in later periods (>5 years) of the disease is a serious problem in breast cancer. Breast cancer tissues from a large cohort in Japan were evaluated by conventional immunohistochemical methods for the presence of the molecules involved in the RB1CC1 pathway, including RB1CC1, RB1, p53, and other well-known prognostic markers for breast cancer, such as estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2. The correlation between the immunohistochemical results and clinical outcomes of 323 breast cancer patients was analyzed using a Kaplan-Meier log-rank test and a multivariate Cox proportional hazards regression analysis. Absence of nuclear RB1CC1 expression was associated with the worst prognosis (Log-rank test, Chi-Square value = 17.462, p<0.0001). Dysfunction of either one of RB1CC1, RB1, or p53 was associated with the highest risk for cancer-specific death, especially related to survival lasting more than 5 years (multivariate Cox proportional hazard ratio = 3.951, 95% Confidence Interval =1.566-9.967, p = 0.0036). Our present data demonstrate that the combined evaluation of RB1CC1, RB1 and p53 by conventional immunohistochemical analysis provides an accurate prediction of the long-term prognoses of breast cancer patients, which can be carried out as a routine clinical examination.