Analysing entrainment of cardiac and locomotor rhythms in humans using the surrogate data technique.

Using the surrogate data technique we evaluated whether, during running, the synchronization between cardiac and locomotor rhythms resulted from entrainment or by chance. An electrocardiogram and an electromyogram from the right vastus lateralis muscle were monitored from ten healthy young men running at a paced rhythm of 150 steps a minute. The relationship between cardiac and locomotor rhythms was determined by examination of the occurrence of the heart beat with respect to the locomotor phase. The examination revealed that synchronization patterns were observed in all subjects. We generated surrogate data by sorting randomly the original locomotor rhythm, and no synchronization patterns were then seen. This may indicate that the synchronization between the cardiac and locomotor rhythms represented entrainment. We have provided the first evidence for the rejection of the hypothesis that when heart beat rhythm is close to the locomotor rhythm, synchronization between the two rhythms occurs by chance.

Osteogenesis coordinated in C3H10T1/2 cells by adipogenesis-dependent BMP-2 expression system.

A novel tissue engineering for bone formation has been proposed, to make osteoblast differentiation balanced by transfecting the mesenchymal stem cells with a gene encoding human bone morphogenetic protein-2 (hBMP-2) under the control of adipocyte specific lipoprotein lipase (LPL) promoter. Due to the promoter specificity, the initiation of BMP transcription is dependent on adipogenesis. For 14-day culture in the presence of ascorbic acid (asc) and beta-glycerophosphate (gly), nontransfected mouse embryonic fibroblast C3H10T1/2 (10T1/2) cells showed extensive accumulation of lipid droplets and adipocyte specific enzyme glycerol-3-phosphate dehydrogenase (G3PDH) mRNA expression, but exhibited neither BMP-2 expression, high alkaline phosphatase (ALP) activity which reflects osteoblast phenotype. On the other hand, transfected 10T1/2 cells showed hBMP-2 expression, high ALP activity and low level of G3PDH. mRNA expression accompanied with minimal lipid droplets. These results indicate that 10T1/2 cells are proved to be differentiated with maintaining coordinated balance of adipogenesis and osteogenesis, when they are transfected by the gene encoding hBMP-2 under the control of LPL promoter.

Genetically fused protein A-luciferase for immunological blotting analyses.

The gene expression plasmid pMALU5 for the fusion protein of protein A (SpA) with a complete sequence of firefly luciferase (Luc) was constructed. The fused gene was expressed in Escherichia coli, and the resulting SpA-Luc fusion protein was purified by one-step affinity chromatography on IgG-Sepharose. The protein retained both activities: IgG binding capability of protein A and enzymatic activity of luciferase. Blotting analyses were performed with the fusion protein to determine a tumor marker of alpha-fetoprotein (AFP). AFP was detected at the lowest detection limit of 5 pg by dot blotting and Western blotting. The SpA-Luc fusion protein provides a highly selective, sensitive, and versatile marker for blotting analyses.

Electrically stimulated induction of hsp70 gene expression in mouse astroglia and fibroblast cells.

Mouse astroglial cells, which were cultured on an electrode, were found responsive to an electric stimulation of sine wave potential in enhancing hsp70 mRNA resulting from an activation of hsp70 gene expression. On the basis of this finding, electrically responsive cells were established by transfecting mouse 3T3-L1 cells with a constructed plasmid encoding hsp70 promoter and the firefly luciferase gene. A stable cell line has been established through selection of heat-stimulated luciferase expression. A 1-h electric stimulation of the cells resulted in activation of luciferase expression, which was confirmed to produce an increase in light emission. The sequential pattern of the electrically stimulated expression of luciferase was found different from that of the heat stimulation. Furthermore, the promoter was activated depending on the potential and duration of the stimulation applied. Consequently, the electric stimulation has proven effective on activating hspP70 promoter. This cell line is feasible in expressing the gene of interest by electrical stimulation, which lead us to construct environment responsive cells in general.

Single-molecular AFM probing of specific DNA sequencing using RecA-promoted homologous pairing and strand exchange.

The specific sequence in a linearlized double-stranded DNA target has been identified at a single-molecular level by atomic force microscopy (AFM). This was accomplished using RecA-coated, single-stranded DNA probes which were paired with a specific complementary DNA sequence in a linear double-stranded DNA target by strand-exchange reaction at a homologous sequence site with target DNA. The sites of interaction between the nucleoprotein filaments and the double-stranded DNA targets were directly visualized by AFM in solution containing 4 mM magnesium acetate. Measurements of the position of RecA-coated probes paired to individual target DNA showed that DNA probes specifically paired at their corresponding homologous target sequences. Strand exchange promoted by RecA and the visualization by AFM provided a rapid and efficient way to identify homologous sequence on a single-molecule target DNA.

Assembling of engineered IgG-binding protein on gold surface for highly oriented antibody immobilization.

The B-domain, which is one of IgG-binding domains of staphylococcal protein A, was repeated five times and a cysteine residue was introduced at its C-terminus by a genetic engineering technique. The resulting protein, designated B5C1, retained the same IgG-binding activity as native protein A. The B5C1 was assembled on a gold plate surface by utilizing a strong affinity between thiol of cysteine and a gold surface. IgG-binding activity of B5C1 on a gold surface was much higher than that of physically adsorbed B5, which lacks cysteine residue. Furthermore, antigen-binding activity of immobilized antibody molecules through the use of assembled B5C1 on a gold surface was about 4.3 times higher than that of physically adsorbed antibody molecules. Immobilization of highly oriented antibody molecules was realized with the engineered IgG-binding protein.

Design and gene engineering synthesis of an extremely thermostable protein with biological activity.

A novel strategy for designing and synthesizing extremely thermostable biologically active proteins is proposed. The design concept is based on combining a rigid and extremely hydrophobic peptide unit with a biologically active peptide unit. The cell adhesive peptide sequence, Arg-Gly-Asp (RGD), as a functional peptide unit was incorporated into the elastin-based rigid polyhexapeptide, whose repeating unit is Ala-Pro-Gly-Val-Gly-Val (APGVGV). The designed fusion gene was expressed in E. coli, and the resulting protein, designated ER4, was purified with affinity chromatography. The ER4-coated cell culture plate showed sufficient cell adhesive activity through the RGD sequence on the surface of ER4. The thermostability of ER4 was demonstrated by estimating the remaining cell adhesive activity after autoclaving at 120 degrees C for 20 min, and it retained over 90% of cell adhesive activity compared with native ER4.

Site-directed lipid modification of IgG-binding protein by intracellular bacterial lipoprotein process.

IgG-binding protein was genetically expressed and lipid-modified in a site-directed manner in Escherichia coli. The DNA sequence encoding the signal peptide and the nine N-terminal amino acid residues of the major lipoprotein of E. coli (lpp) was fused to the sequence of B-domain which was one of the IgG binding domains of Staphylococcal Protein A (SpA). The N-terminal cysteine residue of the resulting protein was enzymatically linked with lipids in the bacterial membrane. The lipid-modified protein was translocated at the bacterial membrane in a manner similar to native bacterial lipoprotein, and it was purified with IgG-Sepharose by affinity chromatography. The lipid modified proteins (lppB1 and lppB5) showed a similar IgG binding activity to unmodified proteins, which was estimated by competitive ELISA. Proteoliposomes of lipid modified proteins were prepared in an elegant fashion so that the IgG binding site should be properly oriented on the surface of an individual liposome by anchoring the lipid-tail into the hydrophobic layer of the liposome membrane. As compared with the unmodified one, the lipid modified protein incorporated into the proteoliposome exhibited higher IgG binding activity.

In vitro selective RNA synthesis with L-A virus nanoparticles.

New in vitro RNA synthesis has been performed with an L-A virus nanoparticles, in which the gene and polymerase are integrated. The specific recognition sequence (packaging site) of L-A virus was inserted within a gene of interest. Based on the intrinsic replication cycle, the exogenous RNA with the packaging site was encapsulated by an empty L-A virus nanoparticle. The packaging site worked as a recognition site even for exogenous RNAs. The recognized RNA was replicated to dsRNA, and was then transcribed by empty L-A virus nanoparticles. These results indicate that empty L-A virus nanoparticles recognize an exogenous RNA with the packaging site and synthesize RNA in vitro.

Stabilization and translation of immobilized mRNA on latex beads for cell-free protein synthesis system.

The stability of immobilized mRNA against ribonucleases was investigated in a cell-free protein synthesis system. The plasmid-encoding protein A with the 20-mer poly(A) tail under the control of T7 promoter was constructed, and the corresponding mRNA was synthesized by T7 RNA polymerase reaction. The resulting mRNA was immobilized on oligo(dT)-immobilized latex beads by hybridization utilizing the poly(A) tail of mRNA at the 3'-terminus. The mRNA was stabilized against three types of nucleases (3'-OH exonuclease, 5'-OH exonuclease, and endonuclease) by immobilization. Translation of immobilized mRNA with a continuous-flow cell-free protein-synthesizing system from Saccharomyces cerevisiae was ascertained. Reusability of the immobilized mRNA as genetic information was also examined.

Structure and interactions with RNA of the N-terminal UUAG-specific RNA-binding domain of hnRNP D0.

Heterogeneous nuclear ribonucleoprotein (hnRNP) D0 has two ribonucleoprotein (RNP)-type RNA-binding domains (RBDs), each of which can bind solely to the UUAG sequence specifically. The structure of the N-terminal RBD (RBD1) determined by NMR is presented here. It folds into a compact alphabeta structure comprising a four-stranded antiparallel beta-sheet packed against two alpha-helices, which is characteristic of the RNP-type RBDs. Special structural features of RBD1 include N-capping boxes for both alpha-helices, a beta-bulge in the second beta-strand, and an additional short antiparallel beta-sheet coupled with a beta-turn-like structure in a loop. Two hydrogen bonds which restrict the positions of loops were identified. Backbone resonance assignments for RBD1 complexed with r(UUAGGG) revealed that the overall folding is maintained in the complex. The candidate residues involved in the interactions with RNA were identified by chemical shift perturbation analysis. They are located in the central and peripheral regions of the RNA-binding surface composed of the four-stranded beta-sheet, loops, and the C-terminal region. It is suggested that non-specific interactions with RNA are performed by the residues in the central region of the RNA-binding surface, while specific interactions are performed by those in the peripheral regions. It was also found that RBD1 has the ability to inhibit the formation of the quadruplex structure.

Two types of electrochemical nitric oxide (NO) sensing systems with heat-denatured Cyt C and radical scavenger PTIO.

To develop an in situ NO sensing system for primarily biological and medical uses, two types of NO sensing materials, which may be coupled with an electrochemical reaction for signal transduction, have been investigated. Heat-denatured Cyt C and a radical scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetra methyl imidazoline-1-oxyl-3-oxide (C-PTIO) were found to be effective and were incorporated into electrochemical sensing systems. Heat-denatured Cyt C deposited on a 4-mercaptopyridine modified gold electrode responded to NO with an increase of cathodic current through electrochemical reduction of Cyt C (Fe3+), when the electrode potential was controlled at 0 mV vs Ag/AgCl. The dynamic range of the sensing system was 0.5-4 microM. The sensing system with C-PTIO exhibited an anodic output in response to NO at 0.7 V vs Ag/AgCl, and showed a wider dynamic range from 0.05 to 100 microM.

Rapid diagnosis at the outpatient clinic for breast tumors by fine needle aspiration cytology. The utility.

The aim of this study was to emphasize the utility and prove the accuracy of rapid diagnosis at the outpatient clinic for breast tumors by fine needle aspiration cytology [FNAC]. Rapid diagnosis for breast tumors by FNAC is performed on the same day just after mammography and echonography are carried out at our hospital and the result reported to the patients while they are waiting at the outpatient clinic. We evaluated FNAC by rapid diagnosis at the outpatient clinic for 1,786 breast tumors during the last ten years. The cases of no judgement (Class 0) were 11%, negative cases (Class I & II) 72%, suspicious cases (Class III) 7%, and positive cases (Class IV & V) 10%. We experienced only 4 false negative cases and 0 false positive cases among 1,198 cases during the last 5 years, whereas there were 8 false negative cases and 2 false positive cases among 588 cases during the first 5 years. Two false positive cases in the the first 5 years were judged as Class IV, but definitive surgery [mastectomy] was not performed because rapid diagnosis during the operation by frozen section confirmed no malignancy. As a result, all the cases in which mastectomies were performed up to now were confirmed malignant. We emphasize that rapid diagnosis at the outpatient clinic for breast tumors by FNAC is very useful for early detection and treatment and it is very important to consider the histological type of breast tumors by FNAC to prevent misjudgement.

Gene expression in the electrically stimulated differentiation of PC12 cells.

Cell differentiation of PC12 cells was electrically induced to grow neurites in the absence of nerve growth factor (NGF) on the electrode surface, of which potential was modulated by a rectangular wave of potential. The electric stimulation induced the c-fos expression which is essential for cell differentiation. Non-specific calcium channel blocker, lanthanum ion, inhibited the electrically induced differentiation, while NGF-induced differentiation was not suppressed. An L-type calcium channel blocker, nifedipine, also inhibited the electrically induced calcium influx and c-fos expression. Moreover, a stretch-activated (SA) channel blocker, gadolinium ion, inhibited the electrically stimulated differentiation by blocking the calcium influx, but gave no prominent effects on the potassium ion-induced differentiation. Chelerythrine, a specific protein kinase C (PKC) inhibitor, almost inhibited the cell differentiation by the electric stimulation but not by the NGF treatment. These results indicate that the alternative potential may stimulate cell differentiation through a PKC cascade.

Electrically induced neurite outgrowth of PC12 cells on the electrode surface.

Morphological differentiation of PC12 cells cultured on an indium-tin oxide (ITO) electrode has been induced to grow neurites in the absence of nerve growth factor (NGF) by electrical stimulation. Rectangular pulse wave potentials were applied to the electrode at amplitudes of 200 mV and 400 mV with frequencies of 50 Hz, 500 Hz, and 1 kHz. The PC12 cells differentiated most prominently at 200 mV with 100 Hz. No statistically significant differences were observed among the electrically induced neurite lengths. The electrically induced differentiation was completely inhibited by a blockade of calcium influx using LaCl3. This indicates that repeated potential shift in the vicinity of a cellular membrane may stimulate morphological response, probably through calcium ion channels.

A case of microhepatoma associated with schistosomiasis japonica diagnosed by enhanced ultrasonography after hepatic intraarterial injection of carbon dioxide gas.

The patient was a 58-year old man whose complaints were generalized malaise and right epigastralgia. He had liver cirrhosis and schistosomiasis japonica, previously diagnosed by laparoscopy. Computed tomography (CT) showed a high density funicular shadow in the liver. However no tumorous lesions in the liver were visualized. Ultrasonography (US) of the liver showed a reticulate or scaly pattern, but no images of tumorous lesions. Hepatic angiography showed a single, deeply colored image about 1cm in diameter, in the segmentum anterosuperior. Preoperative and intraoperative enhanced US with hepatic intraarterial injection of carbon dioxide gas was performed. It showed a hyperechoic tumor shadow about 1cm in the segmentum anterior. The segmentum anterosuperior including the tumor was partially resected. Pathologically, the tumor was found to be a hepatoma of Edmondson type II, caused by cirrhosis and schistosomiasis japonica. The patient's postoperative course was uneventful. Enhanced US with hepatic intraarterial injection of carbon dioxide gas was useful for the diagnosis and treatment of the microhepatoma associated with schistosomiasis japonica.

[A case report of primary fibrosarcoma originated in the heart invading adjacent organs ].

A rare case of cardiogenic fibrosarcoma is reported. A 66 y.o. male with dyspnea, cough and fatigue was examined. B-mode echo, MRI, and angiography disclosed a large tumor in the left atrium. A large fibrosarcoma and 2 cm of the left atrial wall around the tumor pedicle were removed. The atrial defect and right lower pulmonary vein were repaired with a patch. Four months later, the tumor recurred in the right lung, mediastinum, and left atrium. The tumor, middle and lower lobes of the right lung, and part of the left atrial wall were excised. One year after the first operation, the patient died of air way obstruction due to another recurrence of the fibrosarcoma in the left lung, esophagus, and mediastinum. Cardiogenic fibrosarcoma invading outward for the mediastinal organs is rare.

[Recent advances in the study on the mechanism of brain injury].

A cerebral contusion and DAI (diffuse axonal injury) are practically very important in a medico-legal case of the closed head injury. In this paper, we will report the epitome of the recent advances in the study on the mechanism of them. Coup contusion can be mainly attributed to the skull inbending and/or the skull fracture which develop in the impact region. As to the mechanism of the contrecoup contusion, several theories are reported. During rotational movement of the head, intracerebral shear strains would be produced because of brain lag (Holbourn's rotation theory). Anatomical features of the skull plays an important role (Gurdjian). Relative movement between a brain and a skull induces intracranial cavitation due to pressure gradient (Gross's cavitation theory). Brain is injured by deformation pressure induced by skull deformation and acceleration one done by a movement of the head (Lindenberg). The last one is Courville's transmitted waves of force theory. As to DAI, there is Gennarelli's theory. During a rotational movement of a brain caused by high rate of angular acceleration operating for a long period, intracerebral shear strains occur and injure a brain. As to the brain injuries which include a cerebral contusion and DAI, two theories are reported. Centripetal progression of strains to the core of a brain injuries the brain (Ommaya). Natural frequency of impact determines the nature of resulting injury to the brain (Willinger).

[Fracture of the cervical spine caused by blow in patient with ankylosing spondylitis--a report of an autopsy case].

Fracture of the cervical spine in a patient with ankylosing spondylitis is presented. A 43-year-old male was involved in a fight when drinking. He received blows to his face and the lower jaw, and fell backward on the street and died. The postmortem examination showed abrasions and subcutaneous hemorrhages on the face and the lower jaw. A transverse fracture was observed through the intervertebral disc space between the fifth and sixth cervical vertebrae. The cervical spinal cord was completely ruptured at the fracture site. Ossification of the supporting ligaments and anterior surface of dics were found. The spine was bony ankylosed. The alcohol levels of blood and urine were 2.95 and 3.84 mg/ml, respectively. The cause of death was paralysis of respiration. The victim had suffered from the ankylosing spondylitis for many years. His neck had no mobility. The X-ray films taken at age 42 showed complete ankylosis of the spine, so-called "bamboo" spine. It seemed that the blow to his face and the lower jaw caused hyperextension of the neck and easily caused the cervical fracture because of the loss of flexibility and fragility from osteoporosis in the ankylosed spine.