Immune system phenotyping of radiation and radiation combined injury in outbred mice.

The complexity of a radionuclear event would be immense due to varying levels of radiation exposures and injuries caused by blast-associated trauma. With this scenario in mind, we developed a mouse model to mimic as closely as possible the potential consequences of radiation injury and radiation combined injury (RCI) on survival, immune system phenotype, and immune function. Using a mouse burn injury model and a (137)CsCl source irradiator to induce injuries, we report that the immunological response to radiation combined injury differs significantly from radiation or burn injury alone. Mice that underwent radiation combined injury showed lower injury survival and cecal ligation and puncture (CLP) induced polymicrobial sepsis survival rates than mice with single injuries. As anticipated, radiation exposure caused dose-dependent losses of immune cell subsets. We found B and T cells to be more radiation sensitive, while macrophages, dendritic cells and NK cells were relatively more resistant. However, radiation and radiation combined injury did induce significant increases in the percentages of CD4(+) regulatory T cells (Tregs) and a subset of macrophages that express cell-surface GR-1 (GR-1(+) macrophages). Immune system phenotyping analysis indicated that spleen cells from radiation combined injury mice produced higher levels of proinflammatory cytokines than cells from mice with radiation or burn injury alone, especially at lower dose radiation exposure levels. Interestingly, this enhanced proinflammatory phenotype induced by radiation combined injury persisted for at least 28 days after injury. In total, our data provide baseline information on differences in immune phenotype and function between radiation injury and radiation combined injury in mice. The establishment of this animal model will aid in future testing for therapeutic strategies to mitigate the immune and pathophysiological consequences of radionuclear events.

Detection of autoantibodies against NMDA-type glutamate receptor in a patient with recurrent optic neuritis and transient cerebral lesions.

We describe the case of a Japanese girl with recurrent optic neuritis and transient cerebral lesions. Antibodies against N-methyl-d-aspartate (NMDA)-type glutamate receptors were detected in both serum and cerebrospinal fluid. Results of this case study suggest that the development of autoantibodies against NMDA-type glutamate receptors may play a role in the pathogenesis of central nervous system demyelinating diseases.

Moyamoya syndrome after cranial irradiation for bone marrow transplantation in a patient with acute leukemia.

It is well known that radiation-induced vasculopathy and arteritis are two of the complications of whole brain radiation therapy. Moyamoya syndromes after cranial irradiation among patients with brain tumors were previously reported. However, we could find only three cases of prophylactic cranial irradiation for hematological disorders and no case of cranial irradiation before bone marrow transplantation in patients with acute leukemia. We recently treated a boy who developed moyamoya vessels 1.5 years after cranial irradiation for bone marrow transplantation for acute leukemia. This is the first report of moyamoya syndrome after cranial irradiation for bone marrow transplantation. The mechanism and incidence of vasculopathy after cranial irradiation are unclear. It would be useful to accumulate data and reveal the etiology of moyamoya vessels formation after cranial irradiation.

Differential diagnosis of neonatal mild hypergalactosaemia detected by mass screening: clinical significance of portal vein imaging.

The aetiology of hypergalactosaemia in 100 neonates detected by screening using the Paigen method is discussed. Hypergalactosaemia was transient in 94 cases and persistent in 6. The aetiology among transient cases was unknown in 55, delayed closure of the ductus venosus in 19, heterozygous UDP-galactose 4-epimerase (GALE) deficiency in 16, and heterozygous galactose-1-phosphate uridyltransferase (GALT) deficiency in 6. The aetiology among persistent cases was hepatic haemangioendothelioma with portovenous shunting in 2, and patent ductus venosus with hypoplasia of the intrahepatic portal vein, citrin deficiency, homozygous GALE deficiency, and heterozygous GALE deficiency in one patient each. The abnormalities of the portal system were identified ultrasonographically at the initial consultation and measurements of the total bile acid and alpha-fetoprotein concentrations were helpful in resolving the differential diagnosis. The causes of hypergalactosaemia varied, but a major cause was portosystemic shunt. Evaluation of patients with hypergalactosaemia should not be limited to enzymatic analysis, but should also include hepatic imaging, especially ultrasonography. Additionally, determination of total bile acids and alpha-fetoprotein is helpful in identifying the aetiology of hypergalactosaemia in infants.

A simple and rapid enzymatic assay for the branched-chain alpha-ketoacid dehydrogenase complex using high-performance liquid chromatography.

Maple syrup urine disease (MSUD) is caused by a congenital defect of the branched-chain alpha-ketoacid dehydrogenase complex (BCKADC), and is one of the target disorders in newborn screening. However, it is not always easy to confirm the diagnosis; conventional methods of enzyme assay require cell culture, isolation of mitochondria, or radioisotope-labelled reagents, and disease-causing mutations can exist in any of the genes encoding the three enzyme subunits. To realize a practical test for diagnostic confirmation, we developed a simple and rapid enzymatic assay for BCKADC. In this procedure, the production of isovaleryl-CoA from 2-ketoisocaproic acid was measured using high-performance liquid chromatography. Detection of the BCKADC product was significantly reproducible depending on concentration of the substrates. We applied the assay to two patients with MSUD and demonstrated pathologically low levels of residual activity in both subjects. These results indicate that our method is a practical and sensitive assay for BCKADC, and that it can be a useful adjunct in newborn screening for MSUD.

The Ca(2+)-ATPase of the scallop sarcoplasmic reticulum is of a cold-adapted type.

At 0 to 20 degrees C, the Ca(2+)-ATPase activity of the scallop sarcoplasmic reticulum (SR) was observed to be 7-60% of the peak activity at 30 degrees C, while the ATPase activity of the rabbit SR was 0-7% of its peak at 55 degrees C. The relative rabbit ATPase activity (0.7-7.0%) at 7-20 degrees C became higher (6-15 times) and lower (1/4-1/2), respectively, by the solubilization of the rabbit ATPase with a detergent, dodecyloctaethylenglycol monoether, and by the reconstitution of the ATPase with asolectin (soybean lecithin). No activity at 0 degrees C remained irrespective of these treatments. The relative scallop ATPase activity at 0-20 degrees C was, however, scarcely affected by such solubilization and reconstitution. In contrast to the rabbit ATPase, the scallop ATPase seems to be capable of operating independently without the help of the membrane lipid at low temperature.

Total endoscopic parathyroidectomy.

During the last five years, minimally invasive procedures have been adopted for the surgical treatment of hyperparathyroidism, because preoperative localization studies such as high-resolution ultrasonography and sestamibi scintigraphy, guidance by intraoperative scans, and the use of quick, intraoperative parathyroid hormone assay have improved. Endoscopic parathyroidectomy was performed by Gagner in 1996, and surgical procedures using endoscopy have been devised. The endoscopic procedures range from the "pure" endoscopic approach characterized by constant gas insufflation to video-assisted gasless techniques. We adopted the "pure" endoscopic approach, because a small incision can be made far from the neck and the cosmetic result is excellent. We report our technique with no scars in the neck region for endoscopic unilateral neck exploration with primary hyperparathyroidism and for endoscopic bilateral neck exploration with renal hyperparathyroidism.

Intraoperative quick parathyroid hormone assay in the surgical management of hyperparathyroidism.

Intraoperative quick parathyroid hormone (QPTH) assay is claimed to prevent failure during parathyroidectomy for hyperparathyroidism. The causes of operative failure have included multiglandular disease, ectopic parathyroid glands, supernumerary parathyroid glands, errors in frozen section evaluations, and missed diagnosis. A QPTH assay has been recognized as a useful method of determining whether hyperfunctioning tissues have been completely excised. However, an intraoperative QPTH assay may fail to detect the presence of double parathyroid adenomas. Use of this assay in conjunction with preoperative and intraoperative localization studies has led to the advocacy of more directed cervical procedures, such as limited, video-assisted, and endoscopic parathyroidectomy.

Minimally invasive radioguided parathyroidectomy.

The recent development of radioguided parathyroidectomy has allowed parathyroidectomy to be quickly performed by a significantly less invasive procedure. Radioguided parathyroidectomy is also likely to decrease operation time, risk of complications, hospital stay, and the overall cost of patient care, result in a smaller scar and rapid, nearly pain-free recovery, and allow local anesthesia. Despite these apparent benefits, a case-control study showed no distinct advantages of employing intraoperative sestamibi identification during parathyroidectomy.

Total endoscopic thyroidectomy: axillary or anterior chest approach.

We have developed endoscopic thyroidectomy by an anterior chest approach and an axillary approach. In this study, we evaluate the efficacy of these two types of endoscopic procedures and conventional open surgery. The degree of surgical invasiveness and the nature of patients' complaints after surgery were compared using the results of the operation and a questionnaire. The mean operating time for the endoscopic procedure was significantly longer than that for open surgery; however, there was no difference in postoperative pain in the three groups. Three months after surgery, the incidence of discomfort while swallowing in open surgery was higher than that in endoscopic surgery. All the patients who were treated using the axillary approach were satisfied with the cosmetic results. However, five patients (25%) who were treated using the anterior chest approach and 15 patients (75%; P < 0.01) who underwent open surgery complained abut the cosmetic results. The incidence of postoperative complaints after endoscopic surgery is significantly lower than that after open surgery. Patients who were treated using the axillary approach can obtain superior cosmetic results, compared with those who received other procedures.

Sentinel lymph node biopsy in patients with thyroid carcinoma.

The subject of performing prophylactic or elective modified dissections of the neck in patients with clinically occult lymph nodal metastases from thyroid cancers is controversial since metastases to lymph nodes are associated with high rates of recurrences. Biopsies of sentinel lymph nodes can be performed successfully in patients with thyroid cancers since they can be identified with dyes or 99mTc. Our preliminary findings indicate that biopsies of sentinel lymph nodes can be useful in the treatment of thyroid cancers; however, the clinical significance of identifying metastases in regional lymph nodes by this technique remains to be determined.

Laparoscopic partial adrenalectomy.

Since corticosteroids are indispensable hormones, partial or cortical-sparing adrenalectomies may be adopted for the surgical treatment of adrenal diseases. In this article, we describe the technique and results of these procedures. Laparoscopic partial or cortical-sparing adrenalectomy has been performed in 10 patients. Seven cases had an aldosterone-producing adenoma (APA) and three had a pheochromocytoma. Three cases with an APA and a case with a pheochromocytoma had tumors located far from the adrenal central vein, and the vein could be preserved. Four cases with an APA and two with a pheochromocytoma had tumors located close to the adrenal central vein, and it was necessary to section the central vein to resect them. All endoscopic procedures were performed successfully. There were no postoperative complications. At follow-up, adrenal 131I-adosterol scintigrams showed the preservation of remnant adrenal function in all patients. Laparoscopic partial or cortical-sparing adrenal surgery was safely performed, and adrenal function was preserved irrespective of whether the adrenal central vein could be preserved or not. We consider this to be a useful operative technique for selected cases.

[Current status of adjuvant endocrine therapy for hormone responsive breast cancer].

Three important meetings on adjuvant hormone therapy for breast cancer were held recently: the 5th EBCTCG Meeting, NIH Consensus Meeting, 7th International Conference on Adjuvant Therapy of Primary Breast Cancer. The conclusions of these meetings are: 1. adjuvant hormone therapy should be indicated only for patients with estrogen/progesterone receptor positive cancer, 2. five years of tamoxifen is the standard care at present, 3. ovarian ablation by any means has been proved effective in premenopausal patients and LH-RH agonist should be given at least two years, and 4. aromatase inhibitors should not be used in clinical practice, because several prospective randomized trials are ongoing at present. The patients treated with LH-RH agonist combined with tamoxifen showed better relapse-free survival compared with LH-RH agonist alone in the INT-0101 trial. This was an important trial because combined hormone therapy had not been proven more effective than individual hormone therapy previously. Combined hormone therapy including LH-RH agonist may be considered in premenopausal patients. There is a growing consensus that chemotherapy is effective through the ovarian suppression. In this sense, hormonal therapy should be considered first for hormone responsive patients. On the contrary, standard chemotherapy has shifted from CMF combination to an anthracycline containing regimen. Chemoendocrine therapy may be considered in high risk patients.

cDNA cloning and predicted primary structure of scallop sarcoplasmic reticulum Ca(2+)-ATPase.

Sarcoplasmic reticulum (SR) Ca(2+)-ATPase of the scallop cross-striated adductor muscle was purified with deoxycholate and digested with lysyl endopeptidase for sequencing of the digested fragments. Overlapping cDNA clones of the ATPase were isolated by screening the cDNA library with an RT-PCR product as a hybridization probe, which encodes the partial amino acid sequence of the ATPase. The predicted amino acid sequence of the ATPase contained all the partial sequences determined with the proteolytic fragments and consisted of the 993 residues with approximately 70% overall sequence similarity to those of the SR ATPases from rabbit fast-twitch and slow-twitch muscles. An outline of the structure of the scallop ATPase molecule is predicted to mainly consist of ten transmembrane and five 'stalk' domains with two large cytoplasmic regions as observed with the rabbit ATPase molecules. The sequence relationship between scallop and other sarco/endoplasmic reticulum-type Ca(2+)-ATPases is discussed.

Dedifferentiated chordoma: a case report.

Dedifferentiated chordoma, i.e. chordoma with a sarcomatous component, is a rare bone tumor. This case report is of a dedifferentiated chordoma in the sacrococcygeal region in a 50-year-old male. The patient was initially diagnosed as having a leiomyosarcoma at the first medical consultation, but after the tumor recurred it was proven to be a dedifferentiated chordoma. The primary tumor, which measured 7 x 5 cm, was located in front of the sacrum so as to compress the rectum forward. The resected specimen showed histological features of chordoma, but the presence in some parts of the tumor of spindle-shaped epithelial cells led to a misdiagnosis of leiomyosarcoma. Thirty-six months later, a local recurrent tumor measuring 24 x 17 cm was identified and promptly resected. The recurrent tumor showed features of pleomorphic cell sarcoma mimicking malignant fibrous histiocytoma. In the pleomorphic sarcomatous area, many mitotic figures were seen, and the MIB-1 labeling index was greater than 40%, which was higher than that of the conventional chordoma area. From histological and immunohistochemical examination, the resected tumor was proven to be a dedifferentiated chordoma. This case highlights the importance of careful study of suspected chordoma to allow early identification of sarcomatous components. When we encounter a chordoma with a spindled epithelial component, we need to distinguish this tumor from a dedifferentiated chordoma and other spindle cell sarcomas such as leiomyosarcoma.

Independence of two conformations of sarcoplasmic reticulum Ca2+-ATPase molecules in hydrolyzing acetyl phosphate. A two-pair model of the ATPase structural unit.

The sarcoplasmic reticulum Ca2+-ATPase molecules have been shown to exist in two conformations (A and B) that result from intermolecular interaction of ATPase molecules (Nakamura, J., and Tajima, G. (1995) J. Biol. Chem. 270, 17350-17354). The A form binds two calcium ions noncooperatively, whereas the B form binds the calcium ions cooperatively. Here, we examined the independence of these two forms in the calcium-activated hydrolysis of acetyl phosphate (AcP) under asynchronous and synchronous conditions of their E1-E2 transitions at 0-5 and 25 degrees C. Irrespective of their synchronism and temperature, the two forms hydrolyzed AcP due to calcium that was bound to each of the forms, indicating the independence of the two forms in hydrolyzing AcP. Taking into account the monomer-dimer transition of the ATPase molecules on the sarcoplasmic reticulum membrane accompanying E1-E2 transition of the molecules (Dux, L., Taylor, K. A., Ting-Beall, H. P., and Martonosi, A. (1985) J. Biol. Chem. 260, 11730-11743), the two types of molecules seem to independently carry out such monomer-dimer transition of the same type of molecules. Two pairs, each consisting of the same type of molecules, are suggested to be the structural unit of the ATPase molecules.

Negative or positive cooperation in calcium binding to detergent-solubilized ATPase of the sarcoplasmic reticulum. Its modulation by a high concentration of ATP.

Two different conformations of chemically equivalent Ca(2+)-ATPase molecules in the sarcoplasmic reticulum have been shown to non- and positive cooperatively bind two calcium ions, respectively (Nakamura, J. (1994) J. Biol. Chem. 269, 30822-30827). At pH 7.40, these ATPase molecules split into E1 (high affinity state for calcium), and E2 (low affinity state for calcium), respectively, before calcium binding. At this pH, calcium binding to the monomeric ATPase, solubilized with dodecyloctaethylenglycol monoether, was studied by examining 45Ca2+ binding to the ATPase and calcium dependence of its phosphorylation, fluorescence intensity, ATP-hydrolysis at a low (5 microM) concentration of ATP, and acetyl phosphate hydrolysis. The results suggest that the solubilized ATPase molecules predominantly preexist in E2 and negative cooperatively (the Hill value (nH) = 0.5-0.6) bind 2 mol of calcium/mol of the ATPase with an apparent calcium affinity (K0.5) of 3-5 microM. The nonequivalences of calcium bindings at the membranous ATPase molecules seem to result from the intermolecular interaction of the molecules. A high concentration (5 mM) of ATP modulated the binding manner so that it became positively cooperative (nH approximately 2) and increased the K0.5 to 0.1 microM.

Decomposition of hydrogen peroxide by metmyoglobin: a cyclic formation of the ferryl intermediate.

1. Metmyoglobin reacted with H2O2 to form ferryl-myoglobin, which reverted back spontaneously to the met-form. 2. Through this cyclic reaction of myoglobin between metMb(III) and ferryl-Mb(IV), we proposed that H2O2, one of the potent oxidants in vivo, can be decomposed continuously in cardiac and skeletal muscle tissues in the absence of catalase and peroxidase.

Hydrogen peroxide plays a key role in the oxidation reaction of myoglobin by molecular oxygen. A computer simulation.

The stability properties of the iron(II)-dioxygen bond in myoglobin and hemoglobin are of particular importance, because both proteins are oxidized easily to the ferric met-form, which cannot be oxygenated and is therefore physiologically inactive. In this paper, we have formulated all the possible pathways leading to the oxidation of myoglobin to metmyoglobin with each required rate constant in 0.1 M buffer (pH 7.0) at 25 degrees C, and have set up six rate equations for the elementary processes going on in a simultaneous way. By using the Runge-Kutta method to solve these differential equations, the concentration progress curves were then displayed for all the reactive species involved. In this complex reaction, the primary event was the autoxidation of MbO2 to metMb with generation of the superoxide anion, this anion being converted immediately and almost completely into H2O2 by the spontaneous dismutation. Under air-saturated conditions (PO2 = 150 Torr), the H2O2 produced was decomposed mostly by the metMb resulting from the autoxidation of MbO2. At lower pressures of O2, however, H2O2 can act as the most potent oxidant of the deoxyMb, which increases with decreasing O2 pressures, so that there appeared a well defined maximum rate in the formation of metMb at approximately 5 Torr of oxygen. Such examinations with the aid of a computer provide us, for the first time, with a full picture of the oxidation reaction of myoglobin as a function of oxygen pressures. These results also seem to be of primary importance from a point of view of clinical biochemistry of the oxygen supply, as well as of pathophysiology of ischemia, in red muscles such as cardiac and skeletal muscle tissues.