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Regulation of the kinetics of intracellular Ca(2+) signals with a novel, membrane-penetrable, inositol 1,4,5-trisphosphate (InsP(3)) receptor/Ca(2+) channel modulator, 2-amino-ethoxydiphenyl borate (2APB), has been investigated using patch-clamp, whole-cell recording to monitor Ca(2+)-activated Cl(-) currents in single isolated pancreatic acinar cells. 2APB itself fails to evoke a detectable current response but it dramatically changes the kinetics of agonist-induced Ca(2+) release from pulsatile spikes to long-lasting, huge Ca(2+) waves, suggesting that 2APB coordinates local Ca(2+) release to generate global Ca(2+) signals. The regulation by 2APB can be elicited by internal perfusion of InsP(3) in a concentration-dependent manner, indicating that this regulation is not mediated through membrane receptors or G protein signal transduction. The InsP(3) receptor blocker heparin, but not the ryanodine-sensitive receptor blockers ruthenium red or ryanodine, abolishes 2APB-mediated regulation of Ca(2+) release. This results also suggest that 2APB effects are mediated through InsP(3) receptors. 2APB substantially modifies single inward Cl(-) current pulse evoked by the photolytic release of caged InsP(3) but not by caged Ca(2+). These data indicate that 2APB-induced regulation is mediated neither by Ca(2+)-induced Ca(2+) release nor by affecting Cl(-) channel activity directly. We conclude that 2APB regulates the kinetics of intracellular Ca(2+) signals, represented as the change in the Ca(2+) oscillation patterns from brief pulsatile spikes to huge, long-lasting Ca(2+) waves. Moreover, this regulation seems to be mediated through InsP(3)-sensitive Ca(2+) pools. 2APB may act as a novel, useful pharmacological tool to study the genesis of intracellular Ca(2+) signals.

Using the patch-clamp method, we studied the mechanism of depolarization of rat pancreatic beta-cells induced by glucagon-like peptide 1 (7-36) amide (GLP-1). GLP-1 caused depolarization in a concentration-dependent manner (0.2-100 nM). Exendin (9-39) amide, a GLP-1 receptor antagonist, prevented the GLP-1-induced depolarization. GLP-1 reduced tolbutamide-sensitive membrane currents evoked by voltage ramps from -90 to -50 mV, recorded in the perforated whole-cell configuration, suggesting that GLP-1 decreased the activity of the ATP-sensitive K+ channel (KATP). This GLP-1 effect was prevented by exendin (9-39) amide. In cells treated with Rp-cAMPS, an inhibitor of the cAMP-dependent protein kinase (PKA), GLP-1 still caused depolarization and reduced the whole-cell membrane current through KATP. Examined in the cell-attached configuration, 20 nM GLP-1, applied out of the patch, had little effect on KATP activity. In the inside-out configuration, the open time probability and the single-channel conductance of KATP in the absence of ATP inside the membrane were unaffected by the presence of 20 nM GLP-1 in the pipette. In both conditions, application of ATP to the inside of the membrane reduced KATP activity. The half-maximal concentrations (ki) of ATP were 11.6 microM without and 5.6 microM with 20 nM GLP-1 in the pipette (P<0.05). The values of the Hill coefficient (h) were 1.03 without and 1.01 with GLP-1. We conclude that GLP-1 reduces KATP activity by elevating the sensitivity of KATP to ATP, resulting in depolarization of pancreatic beta-cells. This GLP-1 action is independent of the cAMP signalling pathway.

Ionic mechanisms and signal transduction underlying noradrenaline (NA)-induced depolarization in single smooth muscle cells of guinea-pig vas deferens were studied. NA caused depolarization followed by action potentials through activation of 1-adrenoceptors. In the presence of nifedipine, no action potential was generated, and the magnitude of the depolarization depended on the concentration of NA (0.1-100 micrometer). NA, through 1-adrenoceptor activation, reduced the magnitude of membrane currents in response to voltage ramp pulses from -90 to -30 mV in a concentration-dependent manner. The reversal potential of the current inhibited by NA changed proportionally to the change in the equilibrium potential of K+, suggesting that NA inhibited K+ channel activity. Treatment of cells with GDPS, an inhibitor of G proteins, or bisindolylmaleimide (BIM), a selective protein kinase C (PKC) inhibitor, prevented the NA inhibition of the currents. Application of 12-O-tetradecanoylphorbol 13-acetate (TPA), an activator of PKC, mimicked the effect of NA. It is suggested that in the smooth muscle of guinea-pig vas deferens, activation of 1-adrenoceptors and the subsequent activation of PKC led to inhibition of K+ currents, which is responsible for the depolarization induced by NA.

Purpose: To clarify the relation between quantitative electroencephalogram (EEG) findings and outcome following corpus callosotomy (CC)., Methods: The degree of bilateral synchrony and morphologic similarity of spike-wave discharges was analyzed by using a cross-correlation analysis and the measurements of amplitude differences between bilateral homologous regions in 22 patients who underwent anterior CCs for intractable symptomatic generalized epilepsies (SGE; 17 patients) and frontal lobe epilepsy (five patients)., Results: Interictal generalized synchronous spike-wave (GSSW) bursts in the SGE patients were disrupted and changed to unilateral spike-waves (USWs) in 11 patients and to bilaterally independent spike-waves (BISWs) in six. The USW group had better surgical outcome than the BISW group. Preoperatively, the USW group had significantly lower interhemispheric synchrony (IS) and fewer regional changes in the side leading in time and the side dominant for amplitude, suggesting unilaterally predominant epileptogenesis that triggered the secondary bilateral synchrony. Postoperatively, the BISW group had a more marked reduction in IS because of independent discharges from bilateral epileptogenic areas, and the USW group had a greater amplitude difference because of unilateralized spike-waves. In addition, an excellent surgical outcome was related to (a) the preoperative degree of the morphologic similarity of the bilateral spike-waves (only a small variation during a burst of spike-waves) and the few instances of regional changes in the side leading in time and in the side dominant for amplitude; and (b) to large postoperative amplitude differences., Conclusion: Preoperative quantitative EEG analyses enabled us to predict the underlying conditions of epileptogenesis and the surgical outcomes in patients undergoing CC.

The effects of corticotropin-releasing factor (CRF) on the intracellular concentration of Ca2+ were studied in isolated single beta-cells of the rat islet. Immunohistochemical staining using CRF-receptor antibodies revealed the presence of both type 1 (CRF-R1) and type 2 (CRF-R2) receptors for CRF in the majority of islet cells. CRF (2 nmol/l) increased cytosolic Ca2+ concentration under 2.8 mmol/l glucose, dependent upon extracellular Ca2+. CRF caused depolarization of the cell membrane, which was followed by action potentials under 2.8 mmol/l glucose. The dose-response relationships of CRF-induced depolarization in the presence of 1 micromol/l nifedipine produced a bell-shaped curve, showing the peak response at 2 nmol/l. In the whole-cell patch-clamp recording, CRF enhanced Ca2+ currents through L-type Ca2+ channels in a dose-dependent manner similar to that for depolarization. In cells pretreated with Rp-deastereomer of adenosine cyclic 3',5'-phosphorothiolate (100 micromol/l), neither depolarization nor an increase in the Ca2+ current was caused by CRF at concentrations <2 nmol/l. In these cells, CRF at 20 nmol/l reduced the Ca2+ current. These results suggest that in single beta-cells of rat islets, CRF, through its own receptor, potentiates Ca2+ influx through the L-type Ca2+ channel by activation of the cAMP/protein kinase A signaling pathway. CRF at a high concentration also shows an inhibitory effect on the Ca2+ current through an unknown signaling pathway.

We report the case of a Japanese girl with a severe type of Moebius syndrome. Her morphological features were a mask-like face, limitation of horizontal eye movements, severe bulbar palsy, multiple and bilateral arthrogryposis including the elbow, knee, and ankle joints, and clubfeet. After birth, her general condition became worse because of repeated apneic spells and aspiration pneumonias due to dysphagia. She finally required tracheotomy. Computed tomography (CT) of the brain revealed minute calcifications on the fourth ventricle floor; this may have been due to severe damage to the brain stem. It is most likely that the various manifestations in our patient were due to disturbance of the blood supply to arteries perfusing the brain stem and to some other arteries, at a critical stage of fetal development.

The whole-cell patch-clamp method was used on A7r5 smooth muscle-derived cell line, and Ba2+ currents through Ca2+ channels were recorded. The A7r5 cells showed voltage-dependent, long-lasting Ba2+ currents which were markedly inhibited by nifedipine (10 microM). The magnitude of the maximum Ba2+ current (IBa(max)) was augmented by an application of dbcAMP (1 mM), but not affected by TPA (80 nM). Noradrenaline (NA) at 100 microM caused an increase in the IBa(max) by 19.7% in the presence of phentolamine (10 microM). This effect was cancelled by Rp-cAMPs (10 microM). In the presence of propranolol (10 microM), NA tended to reduce the IBa(max). Application of Ox-LDLs at 100 microg protein/ml caused an increase in the IBa(max) by 15.7%, whereas native LDLs did not change the IBa(max). Rp-cAMPs was ineffective to the Ox-LDL action on the IBa(max). In the presence of Ox-LDLs, NA augmented the IBa(max) by 21.4% in the presence of phentolamine. These results suggest that Ox-LDLs activate L-type Ca2+ channels of A7r5 cells by a mechanism independent of cAMP/PKA signalling.

Several neurological complications associated with Influenza virus infection are known as a febrile convulsion, polyneuritis, meningitis, encephalomyelitis and encephalopathy. Influenza encephalopathy is the most severe complication associated with Influenza. It may be classified into several subtypes according to the clinical symptoms and laboratory data. In this review, Reye's syndrome, acute necrotizing encephalopathy and hemorrhagic shock and encephalopathy syndrome (HSE) are described in detail. The 1995 influenza pandemia in Nagasaki prefecture was markedly neurovirulent and lethal. Twelve cases developed influenza encephalopathy, and the mortality rate between them was 50%. Almost all of them had never be inoculated of influenza vaccine before.

Preliminary screening of antiviral AIDS drugs has been carried out using three different in vitro assay systems. Among 96 samples of different origin tested, two were shown to inhibit the growth of HIV in vitro. One of the positive samples (plant origin) has hopeful signs, as the ranges of effective doses are wider than those of most of positive samples which had been found by us.

The effect of norepinephrine (NE) was examined on the whole-cell Ba2+ current through L-type Ca2+ channels of freshly isolated smooth muscle cells of guinea-pig vas deferens. The magnitude of maximum Ba2+ current [1Ba(max)] varied in different cells, although the capacitance of the cell membrane was similar (approximately 50 pF). Application of dbcAMP augmented 1Ba(max) by 37%, which was canceled by Rp-cAMPs, while PMA decreased the current by 32%, which was canceled by staurosporine. NE increased 1Ba(max) of the cells which originally showed relatively small 1Ba(max), and decreased the current of the cells which showed larger 1Ba(max). In the presence of phentolamine, NE increased 1Ba(max), and this effect was remarkable in cells showed smaller 1Ba(max). In the presence of propranolol, NE decreased 1Ba(max). The excitatory beta-adrenoceptor activation was canceled by Rp-cAMPs, and the inhibitory alpha-adrenoceptor effect was canceled by staurosporine. It is suggested that NE shows dual (excitatory and inhibitory) actions on the L-type Ca2+ channels of smooth muscle of guinea-pig vas deferens. The excitatory beta-adrenoceptor action mediated through cAMP/PKA is predominant in cells with lower density of the Ca2+ channels, while inhibitory alpha-adrenoceptor action mediated through PKC is predominant in cells with higher channel density.

Modulation of the agonist-specific cytosolic Ca2+ oscillatory pattern by thimerosal has been investigated in single pancreatic acinar cells using patch-clamp perforated whole-cell recording to measure the calcium-dependent chloride current (I(C1)(Ca2+)). 1 microM thimerosal, which fails to evoke Ca2+ oscillation alone, clearly changed the pattern of Ca2+ oscillation from pulsatile spikes (evoked by low concentrations of activators) to sinusoidal or transient oscillations. The mimetic action of thimerosal was independent of extracellular Ca2+, was blocked by extracellular application of dithiothreitol or 10 mM caffeine, as well as by internal perfusion with heparin; but was unaffected by ruthenium red. We conclude that thimerosal modulates the agonist-specific cytosolic Ca2+ oscillatory patterns mediated by sensitizing the InsP3-induced Ca2+ release.

Acetylcholine (ACh) caused repetitive transient Cl- currents activated by intracellular Ca2+ in single rat submandibular grand acinar cells. As the concentration of ACh increased the amplitude and the frequency of the transient Cl- currents increased. These responses occurred also in the absence of extracellular Ca2+ but disappeared after several minutes. Repetitive transient Cl- currents were restored by readmission of Ca2+ to the extracellular solution. The higher the concentration of extracellular Ca2+ readmitted, the larger the amplitude of the transient Cl- currents. Ca2+ entry through a store-coupled pathway was detected by application of Ca2+ to the extracellular solution during a brief cessation of stimulation with ACh. In these experiments too, the higher the concentration of Ca2+, the larger the transient Cl- currents activated by Ca2+ released from the stores. The time course of decrease in total charge movements of repetitive transient responses to ACh with removal of extracellular Ca2+ depended on a decrease in charge movements of each transient event rather than a decrease in frequency of the repetitive events. The decrease of charge movements of each transient event was due to a decrease in its amplitude rather than its duration. The results suggest that in this cell type and amplitude-modulated mechanism is involved in repetitive Ca2+ release and that Ca2+ entry is essential to maintain the repetitive release of Ca2+. The results further suggest that the magnitude of Ca2+ entry determines the number of unitary stores filled with Ca2+ which can synchronously respond to ACh.

Preliminary screening of antiviral AIDS drugs has been carried out using three different in vitro assay systems. Among 138 samples tested, two were found to inhibit the growth of HIV in vitro. Neither of the positive samples has hopeful signs, as the ranges of effective doses of the samples are very narrow.

Preliminary screening of antiviral AIDS drugs has been carried out using three different in vitro assay systems. Among 246 samples of different origin tested, six were shown to inhibit the growth of HIV in vitro. Two of the positive samples have hopeful signs, as the ranges of effective doses are wider than those of most of positive samples which had been found by us.

The influence of boiling water treatment on the surface roughness and surface microstructure of set gypsums was investigated. Typical surfaces before and after immersion in boiling water were compared by means of SEM observation, the Knoop hardness test, and a surface roughness test. The surfaces of set gypsums were rougher than that of an acrylic resin plate, and after immersion in boiling water, highly roughened surfaces and thinner crystal bodies were observed on each specimen under SEM. The knoop hardness of set gypsums was considerably lowered after boiling water immersion. That of die stones was the same or lower than set dental stones. The results showed that even brief immersion in boiling water had profound effects on the dental stone cast, resulting in rougher surfaces and lower hardness of set gypsums.

The store-mediated Ca2+ entry was detected in single and cluster of rat submandibular acinar cells by measuring the Ca2+ activated ionic membrane currents. In the cells where intracellular Ca2+ was partly depleted by stimulation with submaximal concentration of acetylcholine (ACh) under a Ca2(+)-free extracellular condition, an employment of external Ca2+ in the absence of ACh caused a sustained increase of the K+ current without affecting the Cl- current. A renewed ACh challenge without external Ca2+ caused repetitive spikes of both K+ and Cl- currents due to the Ca2+ release. SK & F 96365 inhibited the generation of the sustained K+ current and refilling of the Ca2+ store following the Ca2+ readmission. It is suggested that the Ca2+ enters the cell through the store-mediated pathway new the K+ channels and is taken up by the store. Thus, only Ca2+ released from the store can activate both the K+ and Cl- currents.

In order to examine whether Ca2+ entry is directly involved in controlling exocrine secretion, the Ca(2+)-activated Cl- currents were recorded in single and clusters of rat submandibular gland cells using the whole-cell patch-clamp method. Extracellularly applied acetylcholine (ACh, 10 nM) as well as intracellularly applied GTP gamma S and InsP3 caused repetitive transients of the Cl- currents activated by intracellular Ca2+. These responses occurred also in the absence of external Ca2+, but disappeared after several minutes. Readmission of Ca2+ to the extracellular solution restored the repetitive current transients, while introduction of Sr2+ failed to restore the current signals in spite of the presence of Sr2+ entry detected by microfluorimetry. On the other hand, direct application of Sr2+ to the cell inside caused activation of the Cl- currents although less effectively than Ca2+. When Ca2+ was introduced to the extracellular solution during an interruption of ACh stimulation after the ACh-induced depletion of intracellular Ca2+ store, the Cl- current was not elicited. However, a subsequent challenge with ACh at the same concentration in the absence of extracellular Ca2+ caused repetitive transient Cl- currents. The results suggest that in this cell type the stimulated Ca2+ entry does not by itself activate the Cl- currents but activates them indirectly by triggering Ca2+ release from the intracellular Ca2+ store which may take up Ca2+ soon after the Ca2+ entry.

Saccharomyces cerevisiae synthesizes ergosterol via squalene, but squalene is hardly detected in aerobically grown cells. To obtain a stable squalene-accumulating yeast strain, we attempted to disrupt a gene required in the conversion of squalene to ergosterol, by homologous recombination with a short piece of the gene fragment conjugated with an integration plasmid vector carrying the LEU2 gene. Two mutants that required ergosterol at least for fast growth were isolated. In an aerobic cultivation and with ergosterol supplementation, the two mutants accumulated squalene up to 5 mg/g dry cells. Southern hybridization analysis indicated that both mutants had acquired the vector DNA integrated in the same gene, or nearby genes, on chromosome 12.

A 75-year-old female was admitted to our hospital because of fever and hypotension. The peripheral blood showed 400 leukocytes/microliters with 13,000/microliters platelets. Bone marrow puncture revealed that NCC stood at 14,000 with 50.0% blasts. The surface characters of the blasts were CD13+, CD33+, and HLA-DR+, and blood culture tests were positive. Coagulation tests revealed DIC. Based on the foregoing results, hypoplastic leukemia was diagnosed accompanied by sepsis and DIC, and was placed on the concomitant administration of a combination of low dose Ara-C and M-CSF. After 14 days of Ara-C administration and 26 days of M-CSF, her clinical symptoms improved, with the peripheral blood showing a WBC of 2,800/microliters and platelet count of 111,000/microliters. The percentage of myeloblasts decreased to 7.0%. After the administration of Ara-C was suspended for 2 weeks, another course of low dose Ara-C plus M-CSF administration was carried out and the patient achieved full remission. M-CSF stimulates not only the production of monocytes but increases the number of neutrophils and platelets through monocytes. It is also expected that tumoricidal activity may be realized by the activation of monocytes. In this patients, the concomitant administration of M-CSF and low dose Ara-C was remarkably effective in treating hypoplastic leukemia with severe complication. This result suggests that M-CSF will be useful for the treatment of leukemia.

Preliminary screening for antiviral AIDS drugs has been carried out using three different in vitro assay systems. Among 154 samples tested, nine were found to inhibit the growth of HIV in vitro. One of nine has a hopeful sign, as the range of effective doses of the sample is wide.

To determine whether mitochondrial DNA (mtDNA) fragments found within the nucleus are transcribed, we have differentially screened a HeLaTG cDNA library. A clone that hybridized to mtDNA as well as to c-myc was identified. Analysis of the cDNA disclosed that it contained a mtDNA sequence, encoding cytochrome-c oxidase subunit III (coxIII) that was contiguous with and 5' of a c-myc sequence corresponding to part of exon 2 and exon 3. Hybridization of ScaI-digested DNA with a 1.05 kb c-myc probe revealed a unique band in HeLaTG cells, as well as a band common to HeLaTG and 13 other cell types examined. Solution hybridization of HeLaTG RNA with a radiolabeled, single-stranded cDNA probe containing the coxIII-c-myc junction demonstrated a nuclease-resistant band that matched the full length of the junctional cDNA probe. A smaller band that equaled the size of the c-myc portion alone was also detected. Only the smaller band coinciding with the c-myc sequences was protected from nuclease digestion by RNA from other cells. When a radiolabeled probe synthesized in the opposite orientation was used, nuclease-resistant bands equal in length to the coxIII portion of the probe were detected after hybridization with RNA from all cells. These results indicate that insertion of mtDNA fragments into nuclear genes occurs and that subsequent transcription of a 'chimaeric' or 'fusion' mRNA containing both mitochondrial and nuclear sequences can ensue.

Preliminary screening for antiviral AIDS drugs was carried out using three different in vitro assay systems. Among 104 samples tested, six were found to inhibit the growth of HIV in vitro. Four of six were acidic polysaccharides of sea weed origin and one was a well-known anti HIV chemical whose anti-HIV activity has not been reported.

The structural gene encoding human 3 beta-hydroxysteroid dehydrogenase/delta 5----4-isomerase (3 beta HSD) was isolated from a human EMBL3 genomic library. The gene encompasses approximately 8 kilobases of DNA and is comprised of two large introns and three exons encoding amino acid residues 1-48, 49-103, and 104-373, respectively. The exonic sequence is identical to that of the cDNA that we previously isolated and expressed in COS 1 cells. DNA sequence analysis reveals a putative TATA (TATATAA) motif 26 basepairs up-stream of the beginning of exon I, as determined by S1 nuclease protection analysis. However, primer extension analysis using poly(A)+ RNA isolated from both placenta and corpora lutea indicates that the RNA initiates up-stream of the putative TATA motif, and that an additional 53-basepair exon, which is untranslated, is present 5' to the first coding exon. Southern hybridization analysis of genomic DNA using a single exon probe suggests that there may be more than one copy of the gene in the human genome. In addition, we confirm from Southern analysis of genomic DNA isolated from human x hamster somatic cell hybrids that the gene is located on human chromosome 1. These findings will provide a foundation for the characterization of apparent 3 beta HSD clinical deficiencies when these are due to a mutation in the structural gene.

Spinal somatosensory evoked responses to peroneal nerve stimulation were examined in 23 control subjects and 8 patients with pathological lesions of the spinal cord or peripheral nerve. In the control subjects, the response was found as a triphasic potential increasing in latency rostrally at the lumbar spinous recording location. Another negative potential following this triphasic potential appeared at the L1 to T10 spinous recording locations, which might reflect synaptic and/or post-synaptic activity in the spinal cord. This negative response then progressively increased in latency rostrally. The spinal conduction velocity was higher at the upper thoracic leads than at the lower leads. Three patients with spinal cord atrophy showed disappearance of the spinal evoked potential at the spinous recording location corresponding to the pathological lesion. However, since the triphasic potential at the lumbar spinous lead was undetectable in the patients with lesions of the peripheral nerve or cauda equina, the spinal cord function could not be estimated well in these patients.

In 1970 Adams and Kamentsky reported on a technique for classifying human peripheral leukocytes into three groups-lymphocytes, monocytes and granulocytes using supravital staining with a solution of acridine orange, and analyzing the resulting cell suspension with the Cytofluorogram. For the purpose of clinical application of flowcytometry, we investigated the clinical significance of this cytofluorogram of peripheral leukocytes. Cytofluorograms showed characteristic pattern in such hematopoietic malignancies as acute leukemias, chronic leukemias and malignant lymphomas, and turned out to be applicable to the diagnosis of these diseases. Especially in malignant lymphoma, cytofluorogram showed either normal or abnormal pattern. An abnormal cytofluorogram indicated poor prognosis, and an abnormal pattern of cytofluorogram became normalized after chemotherapy against lymphoma. Interestingly, those patients who showed in abnormal pattern relapsed in a short period, even when they were though diagnosed in remission. These data suggest that cytofluorograms are useful in diagnosing, predicting relapse and evaluating therapeutic effects for the treatment of malignant lymphoma. We hope that the analysis of cytofluorogram of peripheral leukocytes in lymphoma will provide a new benefit for further investigation of this disease.

P4 (P14 in adults), N' (N18) and N1' (the negative wave after cortical N1, obtained until late infancy) of short-latency somatosensory evoked potentials (S-SEPs) by median nerve stimulation were studied in 13 selected patients. Of these, 12 patients were suggested to have brain-stem lesions responsible especially for the cardinal symptoms and at the same time showed abnormal S-SEPs characteristically between P3 (P13) and N1 (N20). Another patient showed no cortical response with multilocular cystic encephalopathy. Each wave component could be identified by the same methodology as in controls from neonates to adults. In the normal subject the wave forms of P4 and N' were slightly influenced by sleep level. The abnormal S-SEP findings could be classified by an increase and decrease in the wave amplitudes or by a shortening and prolongation in the peak latencies. Upon these classifications we tried to delineate the pathophysiology underlying abnormalities of S-SEPs between P3 and N1. In addition, a negative wave after N1, compatible with N1', was identified in a case without cortical response. Evidence suggests that the origins of P4 and N' may include not only the lemniscal pathway but partly also the extra-lemniscal synapses in the brain-stem. The neural source of N1' seems to be partly in the subcortical structures. As for the clinical significance of S-SEPs in children, the features of P4 and N' are influenced both by the distribution of the affected lesions and by the underlying pathophysiology.

We isolated from a HeLa genomic library 38 plaques that hybridized to total mitochondrial (mt) DNA isolated from human placenta. One clone (HLmt-17.8) hybridized to a 740 base-pair (12 S ribosomal RNA gene and displacement loop) mtDNA probe and was characterized in more detail. Within its 17.8 x 10(3) base-pair insert a 1.6 x 10(3) base-pair mtDNA fragment was similar to three non-sequential coding genes of human mtDNA, including a part of the 12 S ribosomal RNA (684-971), the cytochrome oxidase I (6553-7302), and two NADH dehydrogenase [ND4L/ND4] (10,606-11,159). The similarity to human mtDNA sequences was 92.0%, 92.3% and 92.4%, respectively, the highest degree of similarity to human mtDNA so far reported. This is also the first report of several adjacent mtDNA-like sequences in cellular chromosomes. The mtDNA-like sequences in HLmt-17.8 was found in the DNAs of human placenta, freshly isolated human leukocytes, foreskin and several human cell lines; but it was not present in other primates or lower organisms. The HLmt-17.8 mtDNA-like region appears to be a pseudogene that transferred into the nucleus in humans more recently than nine million years ago.

We have evaluated the short latency somatosensory evoked potentials (SSEPs) following peroneal and posterior tibial nerve stimulation in 27 normal children and adults, and then applied SSEPs examination following peroneal nerve stimulation to 6 children with neurological deficits. Features of the evoked potentials following peroneal nerve stimulation in normal children were almost similar to those in adults, but we found several points characteristic in children; a higher incidence of evoked potentials and a clearer appearance of "standing potential" at the lower thoracic vertebral level than in adults. Spinal afferent conduction velocity reached at a maximum at 3-4 years of age. The SSEPs following peripheral nerve stimulation in lower extremities are useful in pediatric neurology to determine the level of the spinal lesion, to reveal the distribution and pathophysiology of the spinal dysfunction, and to analyze the process of the disease progression.

This study consisted of 1) molecular deletion analyses in patients with Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) using the entire cDNA for the DMD gene as hybridization probes, 2) RFLP analyses in a large number of Japanese normal women using 11 DMD-linked cloned DNAs as probes, and 3) segregation analyses with these RFLP data in 17 DMD families in which prenatal or carrier diagnosis was required. The deletion study showed that 18 (43%) of 42 male DMD patients had a deletion within the DMD gene, while no detectable deletion was found in 3 BMD patients. These deletions were preferentially observed at the 5' end of the DMD gene, while no deletion was found in the 3' portion of the gene. Of a total of 15 RFLPs detected with the 11 probes, one was a new RFLP (probe/enzyme: P20/MspI). In 6 RFLPs, the allele frequencies in the Japanese were statistically different from those in the Caucasian. Based on the RFLP data combined with the result of the deletion study, an estimated diagnostic rate for prenatal diagnosis and/or carrier detection in the Japanese DMD families was 63%. The real diagnostic rate obtained from the prenatal and carrier diagnoses, which were practically performed in 17 families, corresponded to the estimation. A protocol useful for the diagnosis in Japanese DMD families is presented.

A method to retain subcutaneously the catheter end connected to a new silicone reservoir was developed in intraarterial infusion chemotherapy and applied to 12 patients. Its advantages were discussed. The method can be performed with only minor invasion; in addition, the retained catheter and reservoir needs no aseptic care. Accordingly, it liberates patients from restrictions in the daily life thus far experienced in the conventional method. The intraarterial infusion therapy can be done more easily and without hospitalization by this method. Thus, the method is considered to be quite beneficial in taking care of advanced cancer patients.