Management of refractory chylothorax after pediatric cardiovascular surgery.

We investigated the optimal treatment for refractory chylothorax after pediatric cardiovascular surgery. We retrospectively reviewed the cases of 15 consecutive patients who developed chylothorax after congenital heart surgery performed between December 2004 and November 2010. Among the 15 patients (12 male and 3 female; median age 13.9 months) who developed postoperative chylothorax, 10 recovered with conservative therapy, such as a low-fat diet, medium chain triglyceride-enriched diet, or total parenteral nutrition. Of the remaining 5 patients who underwent surgical treatment followed by conventional therapy, 4 showed improvement, and 1 died from cardiac failure. Surgical treatment was performed at a median of 19 days after diagnosis of chylothorax. Average drainage output of thoracocentesis for the first 5 days before thoracic duct ligation was 33.1 ml/kg/day. Duration of chylous fluid drainage was significantly longer in surgical patients than in patients who recovered with conservative therapy (p < 0.01). Surgical patients tended to be younger with lower body weight. Significant risk factors for surgical intervention were age <4 months, body weight <4 kg, and duration of drainage >10 days. In cases of refractory postoperative chylothorax, surgical therapy such as thoracic duct ligation should be considered when discharge from the drainage tube is >30 ml/kg/day or chylothorax is not improved within 10 days.

Structural and functional analysis of the apoptosis-associated tyrosine kinase (AATYK) family.

Apoptosis-associated tyrosine kinase (AATYK) is a protein kinase that is predominantly expressed in the nervous system and is involved in apoptosis and neurite growth of cerebellar granule cells. In this study, we cloned three new members of the mouse AATYK family, AATYK1B, AATYK2 and AATYK3. AATYK1B is a splicing variant of the previously reported AATYK1 (referred to as AATYK1A hereafter). In comparison with AATYK1A, these three AATYK members were characterized by having an extra N-terminal region that consists of a signal peptide-like sequence and a predicted transmembrane (TM) region, which is followed by a kinase domain and a long C-terminal domain. Both TM-containing AATYK isoforms (AATYK(+)TM: AATYK1B, 2, and 3) and TM-lacking isoform (AATYK(-)TM: AATYK1A) were recovered in membrane fractions, suggesting that AATYK(+)TM and AATYK(-)TM are transmembrane- and peripheral-membrane protein kinases, respectively. AATYK1A was recovered in the soluble fraction when the cells were treated with 2-bromo palmitate, suggesting that AATYK1A associates with membrane via palmitoylation. The kinase domain was highly conserved among all AATYK members and was shown to be catalytically active. Three AATYK family members were predominantly expressed in adult mouse brains with almost similar expression profiles: widespread distribution over the various brain regions, especially in the cerebellum and hippocampus, and up-regulated expression during development of the cerebellum. In cultured cerebellar granule cells, AATYK1 was abundantly localized in both soma and axons, AATYK2 distribution was restricted to soma, and AATYK3 was punctately present over the cells. AATYK1 was concentrated in the central domain of growth cones of dorsal root ganglion neurons. Our results indicate that AATYK family members are brain-dominant and membrane-associated kinases with slightly different distribution patterns in the developing and adult mouse brain, which may be involved in fine regulation of neuronal functions including neurite extension and apoptosis.

Oligo(2'-O-methyl)ribonucleotides. Effective probes for duplex DNA.

To find novel probes for duplex DNA, we prepared four types of triplexes containing a homopurine-homopyrimidine 15-mer duplex DNA, and examined their thermal stabilities (Tm values). The single strand used for triplex formation were a DNA 15-mer having a defined C-T mixed sequence, and its sugar-modified analogs, namely 2'-fluoro DNA, RNA, and 2'-O-methyl RNA. The 2'-O-methyl RNA and the RNA-containing triplexes were similar in their enhanced stabilities at pH 6.1 and, amongst the four triplexes, the 2'-O-methyl was the most stable at pH 5.0. Furthermore, an experiment using a 34-mer duplex DNA suggested that the 2'-O-methyl RNA-triplex was destabilized, mostly as a result of the incorporation of a mismatched triplet, as compared to the DNA triplex counterpart. Thus, 2'-O-methyl RNA can serve as an effective probe for duplex DNA.

Sequence-specific cleavage of RNA by a hybrid ribonuclease H.

Site-specific cleavage of the 22-, 132- and 534-base RNAs by the DNA/protein hybrid RNase H were examined. The 22-base RNA was chemically synthesized, and 132- and 534-base RNAs were prepared by run-off transcription. The hybrid enzyme cleaves these RNAs, which contain a single target sequence, primarily at the unique phosphodiester bond within the target sequence. The hybrid enzyme performs multiple turnovers, and at a substrate/enzyme ratio of 10:1 the RNAs are almost completely cleaved by the hybrid enzyme at 37 degrees C within 1 h. We propose that hybrid RNase H molecules with various oligodeoxyribonucleotides function as RNA restriction enzymes and are useful for structural and functional studies of RNA.

Postnatal differentiation of cell body volumes of spinal motoneurons innervating slow-twitch and fast-twitch muscles.

Distribution of cell body volumes of motoneurons innervating the soleus (slow-twitch or tonic) and medial gastrocnemius (fast-twitch or phasic) muscles was examined in adult cats and a series of kittens ranging from one to 140 days in age. To identify the soleus (Sol) and the medial gastrocnemius (MG) motoneurons, each group of motoneurons was labeled differentially by utilizing retrograde axonal transport of horseradish peroxidase injected into the muscles. It was verified statistically that in the adult cat, the mean cell body volume of the Sol motoneurons was smaller than that of the MG motoneurons. Difference of the mean cell body volume between the Sol and MG motoneurons was found to be significant around the tenth postnatal day. The mean cell body volumes of both Sol and MG motoneurons increased mainly during the third to the seventh week after birth. After this period, increase of the cell body volume was relatively slight and in the fifth month after birth, some of the motoneurons still appeared to be growing. Our findings also suggested that the MG motoneurons may exhibit the adult pattern of distribution of cell body volume in an earlier postnatal stage than do the Sol motoneurons, and that differentiation of motoneurons into the gamma and alpha types may occur earlier than differentiation into the tonic and phasic types.

Localization of masticatory motoneurons in the cat and rat by means of retrograde axonal transport of horseradish peroxidase.

Topographical localization of monotoneurons supplying the masticatory muscles was investigated in the cat and rat, utilizing retrograde axonal transport of horseradish peroxidase. Following injection of horseradish peroxidase in each masticatory muscle, motoneurons labelled with peroxidase were seen to be aggregated into a cluster within the motor nucleus of the trigeminal nerve. Such clusters of peroxidase-motoneurons innervating each masticatory muscle were demarcated more sharply in kittens than in adult animals. The pattern of the nuclear representation of the masticatory muscles was found to be essentially the same in the cat and rat; it could be summarized as follows: The motor nucleus of the trigeminal nerve could be divided cytoarchitectonically into the dorsolateral and ventromedial divisions; the former was seen in almost the whole rostrocaudal extent of the nucleus, while the latter was localized at the levels of caudal two thirds of the nucleus. In the dorsolateral division, the temporal muscle was represented dorsally and dorsomedially, the masseter muscle ventrolaterally, and the pterygoid muscles ventromedially. In the ventromedial division, the anterior digastric muscle was represented dorsomedially, and the mylohyoid muscle ventrolaterally. It was also confirmed that the motoneurons supplying the posterior digastric muscle were localized in the accessory facial nucleus.

The posteromedial ventral nucleus of the thalamus (VPM) of the cat: direct ascending projections to the cytoarchitectonic subdivisions.

The posteromedial ventral nucleus (VPM) of the cat is divided cytoarchitectonically into the magnocellular (VPMmc), lateral parvocellular (VPMpcl), and medial parvocellular (VPMpcm) divisions. Cell bodies of neurons in the VPMpcm are small, while those in the VPMpcl are small to medium-sized. The VPMmc contains large neurons. Direct projections from the lower brain stem structures to each of the three divisions of the VPM were examined by the retrograde horseradish peroxidase (HRP) method. When HRP injection was done into the VPMmc, labeled neurons were mainly located contralaterally in the ventral division of the principal sensory trigeminal nucleus (Vp), in the rostral part of the oral subnucleus in the spinal trigeminal nucleus (Vsp), and in the interpolar subnucleus of the Vsp; a few labeled neurons were also found contralaterally in lamina I of the caudal subnucleus of the Vsp. When HRP injection was restricted to the VPMpcl or VPMpcm, HRP-labeled neurons were mainly observed ipsilaterally, respectively, in the dorsal division of the Vp, or in the parabrachial nucleus (PBN) regions dorsomedial and ventromedial to the brachium conjunctivum. After HRP injection into the parvocellular part of the VPM (VPMpc), labeled neurons were also seen contralaterally in the Vsp, but these were far less numerous than those seen after HRP injections into the VPMmc. Thus, each of the three divisions of the VPM receives main ascending afferent fibers from different brain stem structures; the VPMpcm, VPMpcl, or VPMmc receives afferent fibers, respectively, from the PBN ipsilaterally, from the dorsal division of Vp ipsilaterally, or from the ventral division of the Vp and the Vsp contralaterally.

A cerebello-pulvino-cortical and a retino-pulvino-cortical pathways in the cat as revealed by the use of the anterograde and retrograde transport of horseradish peroxidase.

A cerebello-pulvino-cortical and a retino-pulvino-cortical pathways were revealed in the cat by means of the horseradish peroxidase (HRP) method. The sites of termination of the cerebellofugal and retinofugal fibers in the pulvinar nucleus (Pul) were visualized by the use of the anterograde transport of HRP. The cerebello-pulvinar fibers were found to arise mainly from the parvicellular region of the lateral cerebellar nucleus and to terminate contralaterally in a narrow area at the extreme dorsolateral edge of the Pul at the level of the stereotaxic frontal plane A-7.0. The area of terminal ramification of retino-pulvinar fibers was seen as a thin sheet lying at the extreme lateral edge of the Pul through most of the rostrocaudal extent of the Pul, bilaterally with contralateral predominance. The cerebellorecipient area in the Pul did not seem to overlap with the retinorecipient Pul area; the former appeared to be contiguous ventrolaterally to the latter. The cerebellorecipient and retinorecipient Pul areas were also observed to be connected reciprocally with the cerebral cortical areas; the former was connected with the most posterior part of the area 20, and the latter with the area 19.

A light and electron microscopic study of premotor neurons for the trigeminal motor nucleus.

Premotor neurons sending their axons to the trigeminal motor nucleus were observed in the cat by light and electron microscopy after labeling the neurons retrogradely or anterogradely with horseradish peroxidase (HRP). After HRP injection into the trigeminal motor nucleus, retrogradely labeled neurons were seen most frequently in the parvocellular reticular formation bilaterally. Many labeled neurons were also seen contralaterally in the intermediate zone at the rostralmost levels of the cervical cord and its rostral extension into the caudalmost levels of the medulla oblongata. Additionally, some neurons were labeled ipsilaterally in the mesencephalic trigeminal nucleus, contralaterally in the main sensory trigeminal nucleus and the trigeminal motor nucleus, and bilaterally in the oral and interpolar subnuclei of the spinal trigeminal nucleus. Only a few labeled neurons were seen in the confines of the gigantocellular reticular formation. All labeled neurons were small or of medium size; no large neurons were labeled. After HRP injection into the regions around the trigeminal motor nucleus or the parvocellular reticular formation, axodendritic terminals containing HRP granules were found contralaterally within the trigeminal motor nucleus. Some of these labeled terminals were filled with round synaptic vesicles and others contained pleomorphic synaptic vesicles. The varied morphology of labeled axon terminals was considered to reflect the functional heterogeneity of the premotor neurons for the trigeminal motor nucleus.

Central distribution of afferent and efferent components of the pudendal nerve in cat.

Central distribution of afferent and efferent components of the pudendal nerve was examined in the cat by the HRP method after applying HRP to the central cut end of the pudendal nerve. Retrogradely labeled neuronal cell bodies were located primarily in the feline homologue of the Onuf's X nucleus, constituting a slender longitudinal cell column in the ventral horn of the S1 and S2 cord segments. The Onuf's nucleus was present constantly from middle S1 to high S2 cord segments, and occasionally extended rostrally to high S1 or low L7, and caudally to middle S2, low S2, or high S3 cord segments. No sex differences were observed in the distribution pattern, number, and soma size of labeled neurons in the Onuf's nucleus. Transganglionically labeled dorsal root fibers were found to terminate ipsilaterally in the lamina I of the dorsal horn at levels of lower lumbar, sacral, and higher coccygeal cord segments and the gracile nucleus, and bilaterally with an ipsilateral predominance in the dorsal commissural gray and laminae III, IV, V, and VI of the dorsal horn at levels of lower lumbar, sacral, and higher coccygeal cord segments. Some labeled dorsal root fibers appeared to end ipsilaterally in the regions where the sacral parasympathetic preganglionic neurons have been shown to be located.

A new method for studying the binding of human IgE to CD23 and the inhibition of this binding.

CD23, a low-affinity receptor for IgE (Fc epsilonRII), is a type II membrane-bound glycoprotein expressed on many hematopoietic cells, particularly activated B-cells. CD23 binds to IgE at a domain homologous to Ca2+-dependent (C-type) animal lectin. This paper describes a binding assay by which only the specific binding of IgE to CD23 expressed on Epstein-Barr virus (EBV)-transformed B-cell line, L-KT9 cells, can be detected. This assay is useful in the search for CD23 ligands among many chemical compounds, because it is easily carried out and does not require the use of any radiolabeled reagents. Using the assay, we investigated the inhibition of IgE binding to CD23 by fucose-1-phosphate which has been reported to inhibit the binding of sCD23 to IgE [Delespesse, G., Sarfati, M., Wu, C.Y., Fournier, S., Letellier, M., 1992. The low affinity receptor for IgE. Immunol. Rev. 125, 77.] and the binding of CD23 to CD21 [Pochon, S., Graber, P., Yeager, M., Jansen, K., Bernard, A.R., Aubry, T.-P., Bonnefoy, J.-Y., 1992. Demonstration of second ligand for the low affinity receptor for immunoglobulin E (CD23) using recombinant CD23 reconstituted into flourescent liposomes. J. Exp. Med. 176, 389.]. Although both alpha- and beta-L-fucose-l-phosphate/di(cyclohexylammonium) salt decreased the extent of IgE binding to CD23, the inhibitory effects were not due to alpha- or beta-L-fucose-1-phosphate but to cyclohexylamine. The inhibitory effect of cyclohexylamine was dose dependent and the effect was decreased when inhibition tests were carried out in the presence of a 10-fold excess of IgE. These results suggest that cyclohexylamine specifically interacts with the binding of CD23 and IgE.

A synergistic increase in transplantable peripheral blood stem cells in mice by co-administration of recombinant human interleukin 6 and recombinant human granulocyte colony-stimulating factor.

We examined the effects of co-administration of recombinant human (rh) IL-6 (10 micrograms/day) and rh granulocyte colony-stimulating factor (G-CSF) (0.35 micrograms/day) on the number of peripheral blood cells and peripheral progenitor cells in mice. Among blood cells counts, only white blood cells were synergistically enhanced by co-administration of rhIL-6 and rhG-CSF. Moreover, it was found that co-administration of rhIL-6 and rhG-CSF also caused a marked synergistic increase in the number of peripheral blood progenitor cells. Namely, in combination with rhG-CSF, which alone induced a 170-fold increase in peripheral granulocyte-macrophage colony-forming units (CFU-GM) on day 14, rhIL-6 synergistically increased the number of CFU-GM to more than 1600-fold higher than the number in control mice. Administration of rhIL-6 alone induced a 46-fold increase in CFU-GM. Similar synergistic increases of other hematopoietic progenitors, such as colony-forming units in spleen and megakaryocyte colony-forming units in blood, were also observed in mice co-administered rhIL-6 and rhG-CSF. The survival rate of lethally irradiated recipient mice transplanted with mononuclear cells from 100 microliters of blood from mice administered rhIL-6 and/or rhG-CSF was examined. When mononuclear cells from mice co-administered rhIL-6 and rhG-CSF were injected, survival rate at day 100 was 92%. In contrast, recipient mice transplanted with mononuclear cells from mice administered either rhIL-6 or rhG-CSF alone showed a survival rate of 31% or 46%, respectively, although transplantation of mononuclear cells from control mice failed to rescue any lethally irradiated recipient mice. These results suggest that co-administration of rhIL-6 and rhG-CSF may be useful for peripheral blood stem cell transplantation.

Establishment of high- and low-invasion clones derived for a human tongue squamous-cell carcinoma cell line SAS.

Distant-organ metastasis and regional lymph node metastasis are still the major cause of mortality of oral-cavity squamous-cell cancer (SCC). However, only a few studies have been undertaken to elucidate the mechanism of invasion and metastasis of oral SCC. In this study, we attempted to establish human oral SCC clones with different invasiveness, defined by endothelial cell monolayer assay, which can be used for the study of invasion and metastasis of oral SCC. We established five clones from the human oral SCC cell line SAS by a limiting-dilution method. Two distinct clones, SAS-L1 with very low invasive potential and SAS-H1 with very high invasive potential, were picked out by rat lung endothelial cell monolayer assay. The number of SAS-H1 that penetrated the rat lung endothelial cell monolayer was six fold higher than the number of SAS-L1. There were no differences of metalloproteinase production and cell adhesiveness to Matrigel of SAS-L1 and SAS-H1. However, SAS-H1 exhibited a higher migration ability than SAS-L1. This pair of clones would be a useful experimental model to help in the study of the invasiveness of human oral SCC.

Electron microscopic identification of axon terminals of retinopretectal fibers in the cat by a combined horseradish peroxidase and tritiated amino acids tracing method.

The terminal nucleus of the optic tract in the pretectum was observed electron microscopically in the cat after intravitreous injection of a mixture of HRP and tritriated amino acids. Autoradiographic silver grains, and often also HRP granules, were seen in large axon terminals containing round synaptic vesicles and pale mitochondria. These terminals occasionally made synaptic contracts with the presynaptic dendrites and were involved in the formation of the synaptic triad.

A quantitative electron microscope study of cerebellar axon terminals on the magnocellular red nucleus neurons in the cat.

In the red nucleus (RN) of the cat, the bouton covering ratio (BCR: the ratio of whole somatic surface length and that covered with axon terminals) and the density of axon terminals in contact with somatic profiles (DAST: the number of axosomatic terminals per micron of somatic surface membrane) were calculated in each neuronal somatic profile over 60 micron in diameter. The mean BCR was 61.4 +/- 1.43 (S.E. M.)%. The mean DAST of axosomatic terminals filled with spherical synaptic vesicles (S-terminals) was 27.7 +/- 0.95, and that of terminals with pleomorphic and/or flattened vesicles (F-terminals) was 10.3 +/- 1.12. Subsequently, sequential changes of the BCR and DAST of intact terminals were examined in the RN deafferented from the cerebellorubral fibers. The mean BCR and DAST were decreased most markedly during the survival period of 4-7 days; thus decrease was chiefly due to degeneration of S-terminals (BCR: 16.7 +/- 1.17 %, DAST of S-terminals: 7.1 +/- 1.12, DAST of F-terminals: 5.8 +/- 1.22). In the RN 11-63 days after the operation, both the BCR and DAST tended to re-increase slightly and the majority of the re-increased terminals appeared to be F-terminals. Possible meanings of this re-increase of axosomatic terminals are discussed.

Responses of snake muscle spindles to mechanical and electrical stimulation.

Responses of the sensory ending of snake muscle spindle to mechanical and electrical stimulation were examined. In the short-capsule spindle the dynamic index increases more rapidly with increase in initial muscle length. The threshold muscle length for initiating a long sustained discharge from the short-capsule spindle is significantly higher than that for the long-capsule spindle while the position sensitivity is similar in the two types of spindle. The relation between rate of discharge of the ending and current applied to the impulse initiating site was found to be similar in both types of spindle.

An electron microscope study of synaptic organization in the lateral reticular nucleus of the medulla oblongata in the cat.

Synaptic organization in the lateral reticular nucleus (LRN) was investigated electron microscopically in the cat. The number of synaptic knobs encountered in a survey of 69 somatic profiles cut through the nucleolar plane varied from 0 to 20 per profile. In 5564 mum of cell perimeters analyzed the number of synaptic knobs per 100 mu was 5.2, ranging from 0 to 22 in each somatic profile. About 46% of the axosomatic synaptic knobs were filled with round vesicles, and 54% with pleomorphic ones. Out of 1424 axodendritic synaptic knobs, about 63% were filled with round vesicles, and 37% with pleomorphic ones. After placing lesions in the spinal cord, frontal cortices or red nuclear areas, electron-dense degenerated synaptic knobs were observed in the LRN. In the cats with spinal lesions degenerated knobs with the active zones of the synapses were found both on somatic and dendritic profiles. On the other hand, in the cats with cortical or rubral lesions degenerated knobs with the active zones were encountered only upon dendritic profiles. These degenerated knobs found in the present study never exceeded more than 3% of the total synaptic population in the areas examined. Thus, the axon terminals of fibers arising from the spinal cord, frontal cortices and red nuclear areas constituted only a small fraction of the total axonal endings in the LRN.

SNRK, a member of the SNF1 family, is related to low K(+)-induced apoptosis of cultured rat cerebellar granule neurons.

When cerebellar granule neurons obtained from 11-day-old rats were cultured first in high K(+) medium for 4 days, followed by culture in low K(+) medium, the neurons underwent apoptosis and died. This cell death was prevented by actinomycin D, an inhibitor of RNA synthesis. Commitment time of the protective effect of RNA synthesis inhibition on the cell death was examined by adding actinomycin D at various time points after the switch to the low K(+) medium. More than 50% of the cells died when actinomycin D was added 3 h after changing to the low K(+) medium. To identify what kinds of newly synthesized genes are involved in regulation of the low K(+)-induced death, we performed PCR-based differential subtraction analysis using RNA prepared from the cultured neurons 0 and 3 h after changing to low K(+) medium. We isolated a clone that showed an increase in its mRNA level after changing to the low K(+) medium. This clone encoded the 3' untranslated region of SNRK, a serine/threonine kinase. Tissue distribution analysis showed that the mRNA was expressed mainly in the brain and testis. Developmental analysis in the brain showed that the mRNA expression increased in an age-dependent manner until P28, and was slightly decreased in adults. In situ hybridization analysis showed that the mRNA was expressed throughout the brain. The mRNA was shown to be expressed in neurons by double staining with anti-MAP2 antibody. In addition, anti-N-terminal SNRK antibody stained the nuclei of cultured rat cerebellar granule neurons. These results suggested that SNRK may be involved in regulation of low K(+)-induced apoptosis of cultured cerebellar granule neurons.

Identification of thalamic neurons with vasoactive intestinal polypeptide-like immunoreactivity in the rat.

Cell bodies with vasoactive intestinal polypeptide-like immunoreactivity (VIP-LI) were found in the thalamus of the rat. They were distributed throughout the ventrolateral nucleus (VL) and in the whole extent of the thalamic reticular nucleus (R) except for its most rostral part. On the basis of soma diameters, VIP-LI cells in the VL and R were assumed to be projection neurons.