Differential count and time-course analysis of the cellular composition of coelomocyte aggregate of the Japanese sea cucumber Apostichopus japonicus.

Echinoderms have many types of coelomocytes, which have been known to form aggregates immediately after they are removed from the coelom. To assess the roles that each type of coelomocyte plays in aggregate formation, cellular components of coelomocyte aggregates of the Japanese sea cucumber, Apostichopus japonicus, were investigated. The coelomocytes were tentatively classified into 12 types based on May-Grunwald/Giemsa staining. After the coelom was incubated for 30 min or 6 h, the aggregates were disaggregated completely with 200 mM EDTA. Differential counts of the dissociated cells indicated that the largest component of the aggregates was amoebocytes (67.8%) and the second-largest component of the aggregates incubated 30 min was a type of basophilic granulocyte. In the 6h-incubated aggregates, the fraction of amoebocytes decreased to 59.0%, while that of lymphoid cells significantly increased, which suggests that lymphoid cells participate in late-stage aggregation. After 24-h incubation, only a portion of the aggregated cells could be disaggregated with EDTA. After 48 h, most of the cells could not be detached from the aggregates. Microscopy of frozen sections of the aggregates after 6-h incubation revealed that amoebocytes constructed a mesh-like structure to which other types of cells adhered. After 48 h, the borders of the cells and the intracellular granules were not recognizable. In time-lapse microscopy, the aggregates were observed to move on a glass slide, which suggests that aggregates can "crawl" on the intraluminal surface of the coelom toward, for example, injured regions in the body of the sea cucumber.

Ovarian cavity fluid of the viviparous surfperch Neoditrema ransonnetii suppresses the spontaneous cytotoxic activity of head-kidney leucocytes against xenogeneic targets.

In this study, the effect of ovarian cavity fluid (OCF) from a surfperch Neoditrema ransonnetii on the cytotoxic activity of leucocytes was investigated. In an assay targeting RTG-2, a cell line derived from rainbow trout Oncorhynchus mykiss gonads, leucocytes from both the spleen and head kidney showed spontaneous killing activity. Pre-incubation with OCF significantly suppressed the cytotoxic activity of head-kidney leucocytes towards RTG-2. This suppressive activity was due to the presence of low molecular-mass materials. These results suggest that OCF plays significant roles in pregnancy by its ability to modulate cytotoxicity with maternal leucocytes.

Zona fasciculata 21-hydroxysteroids and precursor-to-product ratios in 21-hydroxylase deficiency: further characterization of classic and non-classic patients and heterozygote carriers.

INTRODUCTION: Although much is known about the increased levels of the 21-hydroxylase substrates 17-hydroxyprogesterone (17OHP) and 21-deoxycortisol (21DF) - the biochemical markers of all forms of 21-hydroxylase deficiency (21OHD), only limited information is available on the zona fasciculata (ZF) products distal to the enzymatic block: 11-deoxycortisol (S), 11-deoxycorticosterone (DOC), and corticosterone (B). OBJECTIVE: To investigate whether basal and post-ACTH levels of S, DOC, and B and the 21-hydroxylase precursor-to-product ratios determined by tandem mass spectrometry preceded by high-performance liquid chromatography separation (liquid chromatography-tandem mass spectrometry) could disclose distinct profiles in genotypically confirmed classic (no.=14) and non-classic (NC) (no.=18) patients, heterozygote carriers (no.=61) and wildtypes (WT) (no.=27) for 21OHD. RESULTS: Salt wasting (SW) and simple virilizing (SV) had higher basal levels of DOC with no further increase in response to ACTH. Stimulated DOC was similar in 21OHD patients and carriers but was reduced as compared to WT. ACTH-stimulated B increased gradually from SW and SV through WT. The post-ACTH 21DF/B ratio was able to detect 92% of the carriers among WT. All NC patients could be detected by post-ACTH 17OHP/DOC and 21DF/B, with no overlap with 21OHD carriers. CONCLUSION: Although 21-hydroxylase is a key enzymatic step in both 17-hydroxy and 17-deoxy pathways of ZF, the reaction is mostly affected in the latter pathway, leading to a significant impairment of B production, which may further characterize the 21OHD subtypes. Also, the precursor-to-product ratios, particularly 21DF/B, can demonstrate the distinctive outline of 21OHD subtypes, including carriers and normal subjects.

Possible immune functions of congerin, a mucosal galectin, in the intestinal lumen of Japanese conger eel.

Congerin, a mucosal galectin of the Japanese conger eel, provides chemical fortification through its agglutinating and opsonizing activity. Congerin is produced in the epidermis, and the epithelia of the oral cavity to the esophagus, but not in the stomach or intestine. We hypothesized that congerin secreted from the upper digestive tract can reach and function in the intestinal lumen. We found that congerin possessed marked resistance against digestion by gastric and enteric enzymes of conger eel. It was not degraded until 6h of incubation with stomach extract or intestinal digestion juice. Western blotting demonstrated that congerin essentially remained in the intestinal mucus. The mucus agglutinated rabbit erythrocytes, and the agglutination was hampered by anti-congerin antibody. Furthermore, congerin could bind to some enteric bacteria. These results support the above hypothesis.

Navigation-guided Ommaya reservoir placement: implications for the treatment of leptomeningeal metastases.

Ommaya reservoirs are commonly used in the diagnosis and management of leptomeningeal metastases (LM) from malignant tumors. The present study investigates the utility of an intraoperative navigation-guided technique for Ommaya reservoir placement. Between March 2004 and December 2005, 85 navigation-guided Ommaya reservoir placements were performed in 77 patients with intracranial malignancies at the Komagome Metropolitan Hospital. Anterior horn puncture and posterior horn puncture were used for 59 and 26 procedures, respectively. A slit ventricle was present in 6 cases. All procedures were performed under assistance from the Medtronic STEALTH STATION TRIA navigation system. Computed tomographic (CT) scans were routinely obtained just after completion of the procedure. Patients diagnosed with LM received subsequent treatment. An Ommaya catheter was applied to the ventricular puncture needle registered in the navigation system and was inserted into the lateral ventricle. Using the real-time "Guidance View", the surgeon was able to verify the catheter position continuously during the procedure. Postoperative CT scan revealed an appropriate catheter position in all except for one case. Complications (catheter malposition) occurred in only one case (complication rate, 1.2%). None of the patients experienced hemorrhage or infection. In conclusion, navigation-guided Ommaya reservoir placement was associated with a very low incidence of complications. This method appears to be safe and effective when employed in patients with intracranial malignancy.

IgM secretion and absorption in the materno-fetal interface of a viviparous teleost, Neoditrema ransonneti (Perciformes; Embiotocidae).

We investigated maternal IgM secretion in the ovary and the absorption of IgM by fetuses in a viviparous fish, Neoditrema ransonneti (Embiotocidae). Serum IgM, whose molecular weight was approx. 820k, was purified by two steps of gel filtration chromatography. Immunohistochemistry and Western blotting revealed that IgM was secreted from the epithelia of the ovigerous lamellae of pregnant females into ovarian cavity fluid. The IgM-secreting activity of ovigerous folds showed notable changes according to the reproductive stage. In fetuses, IgM was absorbed as macromolecules by enterocytes of the hypertrophied hindgut. IgM in the fetal blood was also demonstrated, although its concentration remained low during gestation. These findings suggest that IgM was transported from the maternal tissues to embryos via a unique pathway in N. ransonneti.

FDTD Simulation of Nonlinear Ultrasonic Pulse Propagation in ESWL.

The extracorporeal shock wave lithotripsy (ESWL) has come into wide use due to its non-invasive advantage. However, as for the shock wave propagation in human body in relation to practical lithotripsy, there are a few studies on numerical simulation based on models conforming to practical situations. In this paper we report a numerical method of finite difference time domain (FDTD) for analyzing ultrasonic pulse nonlinear propagation in human body tissues and demonstrated it through an experiment of Reichenberger's ESWL in water.

GAFChromic film dosimetry with a flatbed color scanner for Leksell Gamma Knife therapy.

GAFChromic films MD-55-2 have recently been established widely in industrial, scientific, and medical applications as radiation dosimeters. We applied these films to the dosimetry for Leksell Gamma Knife therapy. We used a flatbed image scanner to take digital images of irradiated MD-55-2, and the data were converted to Red, Green and Blue pixel values. The absorbed dose, as derived from the response curve of the Red pixel value, was consistent with the Leksell Gamma Plan dose planning system, for exposures using collimator sizes, 4 mm, 8 mm, and 14 mm. However, the maximum dose in the exposure of the 18 mm collimator was measured to be about 5% smaller than Gamma Plan due to the density effect of the compound material in the head phantom.

Early prediction of infection after craniotomy for brain tumours.

Reliably predicting infection soon after craniotomies would prevent infection and reduce treatment costs and hospitalization expenses. Therefore we analysed potential risk factors and blood count data after craniotomies for brain tumours in order to predict infection as soon as possible after surgery. We analysed 139 patients who underwent craniotomies for brain tumours from January 1997 to December 2001, and divided them into four categories (Types A to D) according to the following: increase in their white blood counts (WBCs) from Day 0 to Day 1, maximum WBC between Day 0 and Day 2, and maximum c-reactive protein (CRP) between Day 0 and Day 4 after surgery. We evaluated potential risk factors and the blood count data for infections via logistic regression analysis. Type D patients had a significantly higher rate of infection (p = 0.0123) than the other Types, while Type B patients had the lowest rate among the four groups (p = 0.0006). When Type A patients suffered CSF leakages, they had a significantly higher possibility of meningitis (p < 0.0001) or scalp infection (p = 0.012). In those Type A or D patients who were male, more than 70 years old, and suffered from metastases from primary lung cancer lesions, the possibility of pneumonia was significantly higher (p = 0.0178). In conclusion, we are able to predict infection within four days after craniotomies for brain tumours according to standard blood count data and certain risk factors. This possibility allows for improved care and better clinical outcomes in patients that undergo craniotomies for brain tumours.

Fractionation of normal and leukemic T-cells by lectin-affinity column chromatography.

A method for rapid fractionation of normal and leukemic T-cells (Jurkat, RPMI-8402, MOLT-4), using lectin-affinity column chromatography, is described. CNBr-activated Sepharose 6MB was used as a non-mobile phase. The gel was covalently conjugated with Dolichos biflorus agglutinin (DBA) over 24 h. The normal cells were eluted by phosphate buffered saline (Ca(2+) and Mg(2+) free), while the leukemic T-cells, interacting with DBA, were removed by N-acetyl-D-galactosamine or by low-concentrated acetic acid as a mobile phase. The cell fractions were detected spectrophotometrically at 600 nm. The rate of cell elution decreased in the order: normal>leukemic T-cells. The viability and the type of separated T-cell fractions were characterized by flow cytometry, using adequate fluorescent antibodies. The interactions between leukemic T-cells and DBA-saturated Sepharose beads were examined by fluorescent microscopy, using fluorescent isothiocyanate-DBA as a fluorescent marker.

Three-dimensional coherent transfer function for reflection confocal microscopy in the presence of refractive-index mismatch.

The three-dimensional (3-D) coherent transfer function for reflection confocal microscopy of high-numerical-aperture objectives is derived and calculated in the presence of refractive-index mismatch when a laser beam is focused into a medium of refractive index different from its immersion medium. This aberrated coherent transfer function is then used to estimate the readout efficiency of 3-D data bits recorded in a thick medium. It is shown that the readout efficiency of confocal microscopy for 3-D bit data storage is decreased with the focal depth of an objective in a recording medium. However, a high readout efficiency can be maintained if the tube length of a reading objective is linearly altered to compensate for the spherical aberration caused by the refractive-index mismatch.

An implantable power supply with an optically rechargeable lithium battery.

A novel power supply for medical implants has been developed. A wireless near-infrared power transmission recharges a lithium secondary battery in the power supply. A photovoltaic cell array embedded under skin receives near-infrared light through the skin and charges the battery directly powering an implanted device. We have shown that, for a photodiode area of 2.1 cm2, 17 min of near-infrared irradiation at a 810-nm wavelength with a power density of 22 mW/cm2 can send enough energy to allow regular commercial cardiac pacemakers to run for 24 h. The temperature rise of the skin during the light irradiation was 1.4 degrees C.

Galectin containing cells in the skin and mucosal tissues in Japanese conger eel, Conger myriaster: an immunohistochemical study.

Congerin is a beta-galactoside binding lectin (galectin) purified from the skin mucus of the Japanese conger, Conger myriaster. To clarify its tissue distribution and productive cells, several tissue samples including skin, buccal cavity wall, tang, pharynx, gills, esophagus, stomach, intestine, liver, kidney, spleen and ovary of conger were stained immunohistochemically using polyclonal rabbit anti-congerin serum. In the epidermis, a number of club cells were strongly stained. Because no agglutinating activity was detected in plasma, it appears evident that congerin is produced and secreted into mucus by those cells. In addition, congerin-positive club cells were distributed in the mucosal epithelium lining the digestive tract preceding the stomach and in the gills. These findings suggest that congerin participates in innate immunity on the intra- and the extra-body surface of the conger. The putative functions of club cells in fish and their contained lectin are discussed.

A study of reproductive performance in pregnant, IL-2 receptor beta-chain overexpressed transgenic mice.

Relationships between female reproductive performance and uterine natural killer (uNK) cells were investigated in pregnant IL-2 receptor beta-chain overexpressed transgenic (Tg2Rbeta) mice. At 8 days of pregnancy, all fetuses were alive, suggesting that implantation normally occurred in these mice. However, 47% of fetuses were dead at 10 days of pregnancy and at 12 days all fetuses were resorbing, indicating that fetal loss progressed with the advance of pregnancy. The placenta of Tg2Rbeta mice gradually decreased in weight with the advance of pregnancy. At 10 days the placental labyrinth, decidua basalis, and metrial gland in Tg2Rbeta mice were poorly developed, and more uNK cells were found in Tg2Rbeta mice than in the control mice. We propose that Tg2RPbeta mice are the first and interesting model that uNK cells can cause abortion, to clarify the involvement of uNK cell function in female reproductive performance.

Complete amino acid sequences of three proteinase inhibitors from white sword bean (Canavalia gladiata).

Three major serine proteinase inhibitors (SBI-1, -2, and -3) were purified from the seeds of white sword bean (Canavalia gladiata) by FPLC and reversed-phase HPLC. The sequences of these inhibitors were established by automatic Edman degradation and TOF-mass spectrometry. SBI-1, -2, and -3 consisted of 72, 73, and 75 amino acid residues, with molecular masses of 7806.5, 7919.8, and 8163.4, respectively. The sequences of SBI-1 and -2 coincided with those of CLT I and II [Terada et al. (1994) Biosci. Biotech. Biochem., 58, 376-379] except only N- or C-terminal amino acid residues. Analysis of the amino acid sequences showed that the active sites of the inhibitors contained a Lys21-Ser22 against trypsin and Leu48-Ser49 against chymotrypsin, respectively. Further, it became apparent that about seven disulfide bonds were present. These results suggest that sword bean inhibitors are members of the Bowman-Birk proteinase inhibitor family.

Biological activities of the lectin, abrin-a, against human lymphocytes and cultured leukemic cell lines.

The cytoagglutination by abrin-a against human cultured cell lines derived from acute lymphoblastic leukemia (ALL) and human peripheral blood lymphocytes obtained from normal adults and from patients with adult T cell leukemia (ATL) was investigated. Among acute T lymphoblastic leukemia (T-ALL) cell lines, abrin-a showed strong cytoagglutination against relatively differentiated cell lines, such as Jurkat and CCRF-HSB-2. Among acute B lymphoblastic leukemia (B-ALL) cell lines, abrin-a strongly agglutinated an immature cell line, NALM6. In comparison with ALL cell lines, cytoagglutination by abrin-a against normal lymphocytes was weak. Abrin-a showed higher cytoagglutination against lymphocytes derived from ATL than lymphocytes derived from normal adults. In connection with the cytoagglutination, abrin-a-induced cytotoxicity against human cultured leukemic cell lines was evaluated. In proportion to the extent of cytoagglutination, abrin-a induced cytotoxicity in Jurkat, CCRF-HSB-2, MOLT-4, RPMI8402, and BALL-1 as well. Although CCRF-CEM and BALM-1 were both weakly agglutinated by abrin-a, these cell lines were very sensitive to the abrin-a-induced cytotoxicity. NALM6 was strongly agglutinated by abrin-a, but abrin-a exhibited less strong cytotoxicity against this cell line. These results suggest the feasible application of abrin-a as a tool to distinguish the human leukemic cells and its potential for clinical application.

Interaction of the hemolytic lectin, CEL-III, with cultured human leukemic cell lines.

We studied interaction of CEL-III with cultured human leukemic cell lines and lymphocytes from normal adults by evaluating the extent of cytotoxicity and cytoagglutination. Among acute T lymphoblastic leukemia (T-ALL) cell lines, CEL-III displayed increased toxicity against different acute lymphoblastic leukemia (ALL) cell lines as a function of increasing differentiation stage. In the case of acute B lymphoblastic leukemia (B-ALL) cell lines, CEL-III showed strong cytotoxicity against relatively immature cell lines. We found that CEL-III was more toxic for ALL cell lines than leukocytes obtained from peripheral blood of healthy adults. Strong influence of the additional amount of calcium ion on the extent of cytotoxicity was observed. In addition, we describe a new way to evaluate the extent of cytoagglutination in "% of agglutinated cells". These findings make CEL-III a promising candidate in research for lectins which bind to and destroy only the targeted leukemic cells.