Proton NMR studies on ischemic rat brain tissue.

The spin-lattice relaxation time (T1) of water protons and the cross-relaxation time (TIS) between irradiated protein protons and observed water protons were measured in order to study water-macromolecular interactions in ischemic rat brain tissues. Tissues were obtained by bilateral common carotid artery occlusion from stroke-prone spontaneously hypertensive rats. Water, Na, and K contents were measured in ischemic brain tissue at the same time. Water and Na content increased while the TIS value and K content decreased following ischemic insults. The T1 value did not change until 180 min after ischemia had been induced. Changes in the TIS value occurred earlier than changes observed for the T1 value, water, and electrolyte contents. Results indicate that the value of TIS may be useful for detecting cerebral ischemia and that the physical structure of water-macromolecular interaction may be altered soon after ischemic onset in brain tissue.     

Perforin-dependent killing of tumor cells by Vgamma1Vdelta1-bearing T-cells

The T-cell subset expressing Vdelta2 paired primarily with Vgamma2 comprises a majority of gammadelta T-cells in human adult peripheral blood and expands significantly during a variety of infectious diseases. In contrast, the other subset of gammadelta T-cells that express Vdelta1 is rare among circulating T-cells and its function is poorly understood. Here, we show that a Vgamma1Vdelta1(+) T-cell line, 3-D, established from human peripheral blood by immortalization with Herpesvirus saimiri was able to specifically recognize tumor cells, such as K562 cells, and release cytotoxic granules containing perforin for target cell killing. Some tumor cells, including Daudi cells that are known to be susceptible to killing by Vdelta2(+) T-cells, were resistant to 3-D killing, implicating distinct pathways for tumor cell control by Vdelta1(+) and Vdelta2(+) T-cells. The 3-D T-cell receptor (TCR):CD3 complex reconstituted in TCR-deficient Jurkat cells was capable of transmitting signals, evidenced by activation of the interleukin 2 (IL-2) gene following ligation with anti-CD3 antibody, yet the TCR-reconstituted cells failed to produce IL-2 in response to the target cells. Thus, these results raise the possibility that some Vgamma1Vdelta1(+) T-cells could potentially be stimulated and lyse tumor cells via ligation of TCR/CD3-unassociated molecules.     

Biomimetic porous scaffolds with high elasticity made from mineralized collagen--an animal study

Histological investigations of a new hydroxyapatite-collagen composite material were carried out to evaluate its possible suitability as a bone substitute. The three-dimensional scaffolds made from biomimetically mineralized collagen exhibit an interconnecting pore structure and elastic mechanical properties. They were implanted into the subcutaneous tissue and bone defects made in the femur of rats and harvested with the surrounding tissue at 1, 2, 4, 8, and 12 weeks after surgery. The materials implanted in the subcutaneous tissue were covered by fibrous connective tissue with a slight inflammatory response, and many foreign-body giant cells were observed on the surface of the scaffolds. Most of the material implanted in the subcutaneous tissue was resorbed at 8 weeks by phagocytosis. In the bone defects, new bone formation was observed on the surface of the material at 1 week. New bone increased with time, and osteoclasts were seen on the surface of the scaffolds at 2 weeks. Resorption and replacement by new bone of many parts of the materials implanted in the femur were observed by 12 weeks. These responses occurred faster than those of other hydroxyapatite-collagen composites. The results suggested that the new biomimetically mineralized collagen scaffolds were suitable as an implant material for bone-tissue reconstruction.     

Intracellular pH measurement in frog muscle by means of 31P-nuclear magnetic resonance.

The 31P-NMR technique was used for the monitoring of intracellular pH and studying its heterogeneity in the femoral biceps muscle of Rana catesbiana under anaerobic conditions. The value of intracellular pH of fresh muscle calculated from the chemical shift of intracellular inorganic phosphate (P1) was 7.3 on average and the line width of P1 was about 0.2 ppm. As the line width determined by the relaxation mechanism was 0.099 ppm, the P1 signal in fresh muscle was concluded to consist of overlapped narrow components, which indicated the heterogeneity of muscular pH (about 0.2 pH unit). Living muscle showed gradual acidification due to glycolysis and the decrease in heterogeneity. When glycolysis was suppressed by iodoacetic acid, slight alkalization due to the breakdown of creatine phosphate was observed. When the Lohmann reaction was suppressed by 2, 4-dinitro-1-fluorobenzene, rapid acidification accompanied by the appearance of a new acidic component was observed with the onset of ATP decrease. This new component was not detected in the muscle pretreated with glycerol to disrupt the transverse tubules. Therefore, it is likely that this new acidic component originates in the intracellular compartment, and not in the cellular difference.     

Morphogenesis of nonpolypoid colorectal adenomas and early carcinomas assessed by cell proliferation and apoptosis

Nonpolypoid neoplasms, as well as ordinary polypoid tumours, are occasionally found in the colorectum. To clarify whether cell kinetic status affects the macroscopic morphology of colorectal neoplasms, we investigated proliferative indices (PI), apoptotic indices (AI), and the expression of apoptosis-related gene products. We examined 110 colorectal neoplasms comprised of 36 polypoid, 38 flat elevated and 36 depressed tumours. According to WHO’s criteria these tumours consisted of 61 adenomas with low grade dysplasia (LGD), 30 adenomas with high grade dysplasia (HGD) and 19 carcinomas with submucosal invasion. Apoptotic cells were detected by TUNEL staining. Proliferating cells and apoptosis-related gene products were assessed by immunohistochemistry for Ki-67, p53, Bcl-2, and Bax antigens. AI were closely associated with macroscopic morphology in adenomas but not in carcinomas. PI were relatively constant among the three macroscopic types in adenomas and carcinomas. Median AI values of polypoid, flat elevated and depressed tumours were 1.8%, 2.1% and 4.6% for adenomas with LGD, 0.8%, 2.4% and 6.2% for adenomas with HGD and 2.9%, 4.0% and 3.6% for carcinomas, respectively. Overall PI were significantly higher in carcinomas than in adenomas with LGD, whereas AI were not different. Although the incidence of expression was significantly higher in carcinomas for p53 and in adenomas for Bcl-2 than the others, the expression of apoptosis-related gene products (p53, Bcl-2 and Bax) was similar among polypoid, flat elevated and depressed tumours. Macroscopic morphology of colorectal adenomas is determined by the apoptosis not by proliferation, and high apoptosis found in depressed adenomas implies their low net growth. Received: 1 July 1999 / Accepted: 17 January 2000     

Effect of circulatory occlusion on human muscle metabolism during exercise and recovery

To assess muscle metabolism and inorganic phosphate (P_i) peak splitting during exercise, 31-phosphorus nuclear magnetic resonance spectroscopy was performed during ramp incremental and submaximal step exercise with and without circulatory occlusion. Seven healthy men performed calf flexion in a superconducting magnet. There was no P_i splitting during ramp incremental exercise with the circulation present and phosphocreatine (PCr) decreased linearly by 0.07 (SEM 0.01) mmol?·?l^?1?·?s^?1, while exercise with the circulation occluded caused the P_i peak to split into a high and a low pH peak. The rate of PCr decrease during exercise with the circulation occluded was 0.15 (SEM 0.03) mmol?·?l^?1?·?s^?1 which with the efficiency of the adenosine 5′-triphosphate (ATP) hydrolysis reaction corresponded well to the mechanical energy. Both with and without occlusion of the circulation PCr decreased with some time lag which may reflect the consumption of residual oxygen. In submaximal step exercise PCr decreased exponentially at the onset of exercise with the circulation open whereas it decreased linearly by 0.15?mmol?·?l^?1?·?s^?1 when the circulation was occluded. After exercise, occlusion of the circulation was maintained for 1 min more and there was no PCr resynthesis. It is suggested that ATP synthesis was limited by the availability of oxygen.     

Continuous measurements of Na, Li, and Cl in the perfused salivary gland by use of NMR

The present study was undertaken to measure tissue contents of Na, Li, and Cl non-invasively in the isolated perfused organ by nuclear magnetic resonance (NMR) using a broadband tunable probe. The NMR signals of 23Na, 7Li, and 35Cl from the isolated perfused rat mandibular gland were collected continuously and each spectrum was obtained for every 15 or 20 s. The Na concentration in the perfusate was varied by replacement with Li, and the resulting changes were monitored by measuring the signal intensities of the electrolytes. The time constant for Na exchange was slower following complete removal of extracellular Na than following its half replacement, suggesting that the Na extrusion by Na+/K+ ATPase was reduced by lowering the extracellular Na level. The time constant for Li exchange was slower than that for Na exchange. The level of Cl was nearly constant during experiment, except for a very slow increase in Cl, possibly resulting from increasing edema and/or intracellular Li storage.     

31P NMR studies on the isolated perfused mandibular gland of the rat

Phosphorus nuclear magnetic resonance (31P NMR) was used to study energy metabolism in the rat mandibular gland. The gland was isolated, perfused arterially and set in the NMR tube. At rest, 7 resonance peaks were observed and 6 peaks identified from low field as: 1) sugar phosphates (SP) and nucleotide monophosphate (NMP), 2) inorganic phosphate (Pi), 3) creatine phosphate (PCr), 4) gamma-nucleotide triphosphate (NTP) and beta-nucleotide diphosphate (NDP), 5) alpha-NTP, alpha-NDP, NAD+, and NADH, 6) an unknown peak, and 7) beta-NTP. From the results of high performance liquid chromatography (HPLC), NTP consisted mainly of ATP and GTP, and UTP was not detected. The tissue contents of ATP and GTP in the perfused gland were determined by HPLC as 1.86 +/- 0.03 and 0.37 +/- 0.01 mmol/kg wet tissue (S.E., n = 5). From 31P NMR and HPLC data, the tissue levels of creatine phosphate, ADP, and sugar phosphates were estimated as 3.3, 0.4, and 4.2 mmol/kg wet tissue, respectively. The cessation of perfusion decreased the tissue levels of PCr and ATP and increased those of Pi and SP. On the other hand, administration of acetylcholine (1 microM), which is an optimal dose for secretion, decreased PCr and increased Pi but did not change SP. The ATP was unchanged initially and slowly decreased to the lower level during sustained secretion. These findings suggest that a sustained secretion requires more energy from ATP hydrolysis rather than initial secretion.     

Targeted Gene Transfer for Adenocarcinoma Using a Combination of Tumor-specific Antibody and Tissue-specific Promoter

We have developed a highly specific gene transfer method for adenocarcinoma using a monoclonal antibody against tumor-specific antigen coupled with a plasmid containing the carcinoembryonic antigen (CEA)-specific promoter. The chimeric CEA promoter (CC promoter), which contained an enhancer from the immediate early gene of cytomegalovirus and the CEA promoter, achieved 4- to 5-fold higher transgene expression in CEA-producing cells than the original CEA promoter while maintaining CEA specificity. Furthermore, a complex of a monoclonal antibody against Lewis Y antigen (LYA), the CC promoter-containing plasmid and cationic liposomes (DOTAP) achieved specific gene expression in CEA-producing and LYA-positive adenocarcinoma cell lines that was 200-fold more efficient than in CEA-non-producing and LYA-negative cell lines during a short in vitro incubation. This strategy may be applicable for clinical gene therapy.