Kazunori Kitagawa

Polyglutamine protein aggregates are dynamic

Protein aggregation and the formation of inclusion bodies are hallmarks of the cytopathology of neurodegenerative diseases, including Huntingtons disease, Amyotropic lateral sclerosis, Parkinsons disease and Alzheimers disease. The cellular toxicity associated with protein aggregates has been suggested to result from the sequestration of essential proteins that are involved in key cellular events, such as transcription, maintenance of cell shape and motility, protein folding and protein degradation. Here, we use fluorescence imaging of living cells to show that polyglutamine protein aggregates are dynamic structures in which glutamine-rich proteins are tightly associated, but which exhibit distinct biophysical interactions. In contrast, the interaction between wild-type, but not mutant, Hsp70 exhibits rapid kinetics of association and dissociation similar to interactions between Hsp70 and thermally unfolded substrates. These studies provide new insights into the composite organization and formation of protein aggregates and show that molecular chaperones are not sequestered into aggregates, but are instead transiently associated.

Circulating interleukin 6 as a useful marker for predicting postoperative complications

We examined postoperative serial changes in the levels of serum interleukin 6 (IL-6), serum acute phase reactants (APRs) and plasma neutrophil elastase (NE) in patients with various cancers and reviewed these changes in patients who did, and did not, show postoperative complications. Serum IL-6 level was elevated after surgery, peaking on the first postoperative day. Elevation of serum APRs and plasma NE levels also followed. There was a significant correlation between the serum peak level of IL-6 and those of APRs and NE (P less than 0.01). Moreover, there was a significant difference in the serum IL-6 level in patients with and without complications. The relationship between the serum IL-6 greater than 400 pg/ml and the incidence of postoperative complications was also marked. These results suggest that circulating IL-6 is a clinically useful marker for the earliest detection and prediction of postoperative complications.

Allele-specific methylation and expression of an imprinted U2af1-rs1 (SP2) gene.

The mouse U2af1-rs1(SP2) gene, which was cloned by a two-dimensional genome scanning method, is expressed exclusively from the paternally inherited chromosome. This gene has significant similarity to U2AF and located in chromosome 11, of which maternal duplication/paternal deficiency results in a small body. In this report, we cloned genomic U2af1-rs1(SP2) and found its promoter was methylated in a maternal-allele-specific manner. This allelic methylation was not established in parental gametes, but established between 1.5 d.p.c. and 12.5 d.p.c. on the contrary, the allele-specific expression occurred in the two-cell stage when transcription initiates. Absence of the methylation of the upstream region in this stage indicates that methylation is not necessary for inactivation of the expression.

Protease inhibitors (gabexate mesylate and ulinastatin) stimulate intracellular chemiluminescence in human neutrophils

The effect of protease inhibitors on the intracellular production of free radicals was investigated by measuring chemiluminescence (CL) elicited from phagocytosed luminol‐bound microspheres (Lumispheres) in human neutrophils stimulated with formylmethionyl‐leucyl‐phenylalanine (fMLP), interleukin‐8 (IL‐8), phorbol 12‐myristate 13‐acetate, or diacylglycerol. Both gabexate mesylate (Foy) and ulinastatin (Miraclid), urinary trypsin inhibitor, increased intracellular CL in a dose dependent manner. Compared to control buffer without protease inhibitor, gabexate mesylate (322 μg/ml) caused about a 10‐fold increase in intracellular CL in stimulated neutrophils, and ulinastatin (3100 U/ml) a twofold increase in neutrophils stimulated with fMLP or IL‐8. When the protease inhibitors were added to the cell suspension after the phagocytosis of lumispheres, CL responses rapidly increased again to the level which was observed when both protease inhibitors and neutrophil stimulants were incubated simultaneously. In contrast, extracellular release of oxygen metabolites from stimulated neutrophils, assayed by a conventional measurement of luminol‐dependent CL, was reduced by the protease inhibitors in a dose dependent fashion. When luminol‐unbound microspheres were incubated with neutrophils stimulated by fMLP in luminol solution, extracellular CL was almost completely inhibited by gabexate mesylate. These results indicate that the protease inhibitors enhance the generation of intracellular CL and suppress the extracellular release of free radicals.

Epithelial-mesenchymal transformation of a newly established cell line from ovarian adenosarcoma by transforming growth factor-Β1

We report on 2 cell lines (designated OVAK‐I and OVAK‐II) established from an adenosarcoma, a rare variant of a malignant Müllerian mixed tumor, of the ovary. OVAK‐I was not tumorigenic in nude mice and showed no responsiveness to transforming growth factor (TGF)‐B1, whereas OVAK‐II was tumorigenic and had various biological properties. Light‐ and electron‐microscopic studies showed that the morphology of the cells was converted from an epithelial to a mesenchymal form by TGF‐B1. Furthermore, the protein and mRNA expressions of the epithelial marker carcinoembryonic antigen and the mesenchymal marker vimentin were down‐ and up‐regulated, respectively, by TGF‐B1. These findings suggested that TGF‐B1 plays a role in epithelial‐mesenchymal transformation and support the single‐stem‐cell theory which explains the origin of malignant Müllerian mixed tumors: that the epithelial and mesenchymal components of these tumors are of common stem cell origin.

Expression of the pancreatic secretory trypsin inhibitor gene in the liver infected with hepatitis B virus

Pancreatic secretory trypsin inhibitor, an acute phase reactant protein, is expressed in the liver in response to inflammatory cytokines, especially in hepatocellular carcinoma. Northern blots of 25 dissected liver tissues from non-hepatitis, chronic hepatitis and cirrhosis patients revealed that 10 (40%) expressed pancreatic secretory trypsin inhibitor. The expression seemed to be closely associated with hepatitis B viral infection, since among the 11 hepatitis B virus-infected samples, nine (81%) were pancreatic secretory trypsin inhibitor-positive. In contrast, this augmented expression was absent in non-infected livers (0/5; 0%), and rare in those infected with hepatitis C virus (1/9; 11%). There was no significant correlation between the pancreatic secretory trypsin inhibitor expression in the liver and the serum level of glutamic pyruvic transaminase, the hepaplastin test, the 15-min retention rate of indocyanine green, or the histological findings of the liver tissues such as lymphocyte infiltration and pseudolobular formation. Furthermore, we identified an almost three-fold increase in the transactivation of pancreatic secretory trypsin inhibitor gene expression in HepG2 cells after transient transfection with HBV-DNA or the X gene in an expression vector. These results suggest that the induction of pancreatic secretory trypsin inhibitor gene expression in livers with chronic hepatitis and cirrhosis is directly affected by hepatitis B virus.

Enhanced attachment and elastase-releasing capacity of neutrophils after surgery

Perioperative changes in neutrophil attachment level and neutrophil elastase-releasing capacity were examined in patients who underwent surgery for either esophageal or gastric cancer. The neutrophil attachment level was significantly increased in both groups to approximately three times that of the preoperative value. The elastase-releasing capacity of nonstimulated neutrophils was not significantly changed, but it was significantly increased in neutrophils stimulated by N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP). Moreover, both neutrophil attachment and elastase-releasing capacity of FMLP-stimulated neutrophils were more enhanced in patients with postoperative complications than in patients without complications. Peak levels of serum interleukin 6, serum alpha 1 proteinase inhibitor, and plasma neutrophil elastase were also significantly higher in patients with postoperative complications than in patients without. These results suggest that during the postoperative period, neutrophils may be primed and activated in response to inflammatory mediators such as cytokines, and that such an alteration of neutrophil functions may reflect the extent of inflammation and may, at times, be implicated in postoperative complications.

Differential Activation of Polymorphonuclear Leukocytes from Peripheral Blood and Exudate in Surgical Patients

We examined the effects of surgical trauma on polymorphonuclear leukocyte (PMN) chemotaxis, production of phagocytosis-dependent intracellular chemiluminescence (CL) and production of phagocytosis-independent total CL in two groups: group A with esophageal cancer, and group B with gastric cancer. The scale of surgical trauma was quantified by measuring interleukin-6 in plasma and exudate from drainage tubes. We found a significant augmentation of chemotaxis, total CL and intracellular CL in both groups during the post-operative week. In group A, the increments in both chemotaxis and total CL of circulating cells were smaller than those in group B, but there was no significant difference in intracellular CL between the two groups. Exudate PMNs were more chemotactic, but produced smaller amounts of total CL than circulating cells. No significant difference in intracellular CL between exudate and circulating PMNs was detected, indicating that phagocytic activity was not affected. We conclude that severe surgical trauma causes circulating PMNs with high chemotactic activity to migrate to sites of injury, but that preactivated PMNs in exudate examined in vitro produce lower amounts of reactive oxygen metabolites in response to soluble chemoattractants in vitro than cells circulating in plasma.