Rapid detection and differentiation of Newcastle disease virus by real-time PCR with melting-curve analysis

Summary. In order to rapidly detect and differentiate Newcastle disease virus (NDV) isolates, a method based on real-time PCR SYBR Green I melting-curve analysis of the fusion (F) protein gene was developed. The detection limit of real-time PCR was 9 × 10² plasmid copies and was 100 times more sensitive than conventional PCR. Thirty eight reference NDV strains were rapidly identified by their distinctive melting temperatures (T_ms): 89.23 ± 0.27 °C for velogenic strains, 90.17 ± 0.35 °C for pigeon mesogenic strains, 91.25 ± 0.14 °C for two lentogenic strains (B1 and Ishii). No amplification was detected from unrelated RNA samples by this method. This real-time PCR directly detected NDV from infected tissues and eliminated the gel electrophoretic step for analyzing PCR product using ethidium bromide. The total time for a PCR run was less than 1 hour. The results obtained in this study showed that the real-time PCR presented here is a good screening test for the identification of NDV.     

Characterization of the murine interleukin 5 receptor by using a monoclonal antibody

Murine interleukin 5 (IL-5), a lymphokine produced by helper T cells, is involved in the regulation of growth and differentiation of B cells and other hematopoietic cells. The receptor for IL-5 has been identified as two cross-linked complexes on T88-M cells (a murine IL-5-dependent early B cell line). In this study the IL-5 receptor was directly characterized by utilizing an immobilized IL-5 column and a rat monoclonal antibody, designated H7, directed against the IL-5 receptor. H7 completely inhibited specific binding of 35S-labeled IL-5 to T88-M cells, and bound to IL-5-responsive cells, e.g. T88-M, BCL1-B20 (a chronic B-cell leukemia), and MOPC104E (a myeloma), whereas H7 did not bind to IL-5-non-responsive cells, e.g. X5563 (a myeloma), FDC-P1 (an IL-3-dependent line), and MTH (an IL-2-dependent CTLL). H7 could barely bind to T88-M cells in the presence of IL-5, and immunoprecipitated a major band with an Mr of approximately 60 kd from the extract of surface-radioiodinated T88-M cells. The precipitation of this 60 kd molecule was inhibited by the addition of IL-5. Analysis with immobilized IL-5 also revealed that a 60 kd molecule bound specifically to IL-5-coupled beads compared with control beads. Furthermore, no additional molecule with a higher Mr that was recognized by H7 appeared under non-reducing, compared with reducing, conditions. The 60 kd molecule recognized by H7 could be digested with N-glycanase to yield a protein band of approximately 55 kd.(ABSTRACT TRUNCATED AT 250 WORDS)     

Postnatal development of structure and arrangement of tendon cells. A scanning and transmission electron microscope study in the rat calcaneal tendon

The postnatal development of the three-dimensional structure and arrangement of the tendon cells in the calcaneal tendon of rats from 5 to 90 days of age were examined under the scanning and the transmission electron microscope (SEM and TEM). Exposed tendon cells were seen as winged bricks with a stellate profile in a cross section. Their slender perikarya were stacked successively in rows along the long axis of the tendon. The plate-like cytoplasmic processes, which were oriented along the perikaryal long axis, extended radially and joined those of the neighboring cells in rows. Therefore, the tubular channels for collagen fascicles were lined with the chained perikarya and their processes of the tendon cells. The cell rows were usually composed of many cells in developing stages, while short rows of one or two cells were observed in the fully developed stage. The cytoplasmic processes were the primary processes in the lateral extension, and their fine branches formed the secondary processes. The secondary processes were numerous at younger stages, showing a fine meshwork due to their mutual joining in the cross sections. In advanced stages, the meshes were coarse and the secondary processes were also perforated either with grouped fenestrations or large pores. In the fully developed stage, the secondary processes were fragmented on the perikarya, while the primary processes extended in the thin delicate sheets with large perforations. The findings in the present study suggest that the tendon cells are arranged in a three-dimensional network by their mutual joining. The tendon cells of the rat calcaneal tendon may not proliferate very much after birth, but do expand their nursing area in line with normal growth by an elongation of the main primary processes and a reduction of the secondary processes and perikaryal mass. The interfascicular clefts, which were caused by an intervention of either the processes at any developmental stages or the fragmented processes at a certain level of the tendon, may also play a role in the passage of tissue fluid.     

Analgesic action of loperamide,an opioid agonist,and its blocking action on voltage-dependent Ca2+ channels

We investigated the relationship between the antinociceptive effect of the opiate agonist loperamide at the spinal level and its inhibitory effect on calcium influx. Intrathecal administration of loperamide showed a significant antinociceptive effect in the formalin test, which was not prevented by naloxone. On the other hand, no significant effects were observed by nicardipine, an L-type specific blocker, or by BAY K8644, an L-type specific agonist, suggesting no significant role of L-type calcium channels in nociceptive signal transduction. Loperamide suppressed the calcium influx in dorsal root ganglion neurons. As the antinociceptive effect of loperamide was not affected by naloxone or other calcium channel blocking toxins, and loperamide showed a direct inhibitory effect on calcium-influx, the analgesic effect of intrathecally injected loperamide might be due to its blockade of the voltage-dependent calcium channels at the terminals of the primary afferent fibers.     

Prevention of hypertensive crisis with ATP during anesthesia for pheochromcytoma

In the anesthetic management of five patients undergoing excision of pheochromocytoma, adenosine triphosphate (ATP) was used for the purpose of regulating systemic arterial pressure during the period of tumor manipulation. ATP was administered at doses of 0.05–0.4mg/kg/min. Systemic arterial pressure showed a significant decrease from 162 ± 17/103 ± 11mmHg before manipulation to 136 ± 21/81 ± 10mmHg during the manipulation period. The plasma catecholamine levels showed significant increases in this period. Immediately after excision, the systemic arterial pressure was maintained at normal levels (118 ± 13/75 ± 16mmHg) by fluid replacement and discontinuation of ATP administration, subsequently becoming 129 ± 19/79 ± 16mmHg. The heart rate was very stable and tachycardia did not ocurr during the manipulation period. Only one arrhythmic episode ocurred in one patient. The systemic vascular resistance index was significantly lower during the manipulation period than before it. It was therefore considered that ATP was useful as an agent for controlling arterial pressue during the anesthesia for pheochromocytoma.(Murata K, Sodeyama O, Ikeda K et al.: Prevention of hypertensive crisis with ATP during anesthesia for pheochromocytoma. J Anesth 1: 162–167, 1987)     

Quantitative evaluation of regional pulmonary ventilation using PET and nitrogen-13 gas

A new quantitative method, "Simultaneous Exponential Equation method" (SEE), has been developed for the analysis of pulmonary ventilation studies using 13N-labeled nitrogen gas and positron emission computed tomography. This method uses Kety's model assuming insolubility of nitrogen gas in blood or tissues. Activity in poorly ventilated regions does not reach the equilibrium in the so-called equilibrium scan (EQ) performed following 3 or 4 min of washin. Therefore EQ images do not represent lung volume images nor do they provide the initial value of washout phase. Our method corrects for these transient phenomena observed during EQ scan and yields idealistic equilibrium state images (lung volume images) as well as more accurate regional ventilatory time constants than a modified Stewart-Hamilton (A/H) method and tomograms of high resolution.     

The Effects of Gold Kiwifruit Intake Timing with or without Pericarp on Postprandial Blood Glucose Level

The purpose of this study was to examine how gold kiwifruit pericarp (pericarp is defined as the skin of the fruit) consumption and the timing thereof affect the postprandial blood glucose profile. The study was conducted on twelve healthy volunteers (six men and six women). According to our results, the simultaneous intake of gold kiwifruit with bread and the prior intake of gold kiwifruit evidently suppressed the postprandial blood glucose elevation compared with exclusive bread intake. There was no significant difference in postprandial blood glucose changes between the ingestion of gold kiwifruit pericarp and pulp and that of gold kiwifruit pulp only. The highest postprandial blood glucose elevation was suppressed by 27.6% and the area under the blood glucose elevation curve by 29.3%, even with the exclusive ingestion of gold kiwifruit pulp. We predicted that the ingestion of both the pericarp and pulp of gold kiwifruit would reduce the postprandial blood glucose elevation to a greater extent than that of gold kiwifruit pulp only; however, there was no significant difference between the two. These results indicate that gold kiwifruit consumption significantly suppresses the postprandial blood glucose elevation regardless of pericarp presence or absence and the timing of ingestion.     

Enhancement of thermal damage in murine tumors by hydralazine-induced modification of blood flow and oxygen tension

We investigated changes in blood flow in normal muscle and in SCC-VII tumors treated by hyperthermia combined with hydralazine, to evaluate the enhancement of thermal tumor damage by hydralazine. We studied SCC-VII tumor-bearing C3H/He mice. Hydralazine was administered by intraperitoneal injection, and tumors were heated by a water bath. We measured blood flow using the laser Doppler method, and oxygen tension using polarography. The response of tumors to therapy was assessed using a growth delay analysis. In tumors, blood flow and O-2 tension significantly decreased with increasing doses of hydralazine. Compared to tumors treated by hydralazine alone or by hyperthermia alone, tumor blood flow was significantly decreased in the group treated by hyperthermia with hydralazine. In tumors treated by hyperthermia with hydralazine, blood flow was significantly decreased with increasing Hyd doses, heat durations, and temperatures. In normal muscle, no decrease in blood flow was induced by hyperthermia, hydralazine, or their combination. In tumors treated by hyperthermia (43 degrees C, 20 min) with hydralazine, a maximum additional growth delay was observed. Our results suggest that a decrease in tumor blood flow caused by hydralazine plays an important role in enhancement of the hyperthermic antitumor effect.     

Dynamic changes in glucose metabolism of living rat brain slices induced by hypoxia and neurotoxic chemical-loading revealed by positron autoradiography

Fresh rat brain slices were incubated with 2-deoxy-2-[18F]-fluoro-D-glucose ([18F]FDG) in oxygenated Krebs-Ringer solution at 36 degrees C, and serial two-dimensional time-resolved images of [18F]FDG uptake were obtained from these specimens on imaging plates. The fractional rate constant (= k3*) of [18F]FDG proportional to the cerebral glucose metabolic rate (CMRglc) was evaluated by applying the Gjedde-Patlak graphical method to the image data. With hypoxia loading (oxygen deprivation) or glucose metabolism inhibitors acting on oxidative phosphorylation, the k3* value increased dramatically suggesting enhanced glycolysis. After relieving hypoxia < or = 10-min, the k3* value returned to the pre-loading level. In contrast, with > or = 20-min hypoxia only partial or no recovery was observed, indicating that irreversible neuronal damage had been induced. However, after loading with tetrodotoxin (TTX), the k3* value also decreased but returned to the pre-loading level even after 70-min TTX-loading, reflecting a transient inhibition of neuronal activity. This technique provides a new means of quantifying dynamic changes in the regional CMRglc in living brain slices in response to various interventions such as hypoxia and neurotoxic chemical-loading as well as determining the viability and prognosis of brain tissues.