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Despite its charismatic appeal to both scientists and the general public, remarkably little is known about the giant squid Architeuthis, one of the largest of the invertebrates. Although specimens of Architeuthis are becoming more readily available owing to the advancement of deep-sea fishing techniques, considerable controversy exists with regard to topics as varied as their taxonomy, biology and even behaviour. In this study, we have characterized the mitochondrial genome (mitogenome) diversity of 43 Architeuthis samples collected from across the range of the species, in order to use genetic information to provide new and otherwise difficult to obtain insights into the life of this animal. The results show no detectable phylogenetic structure at the mitochondrial level and, furthermore, that the level of nucleotide diversity is exceptionally low. These observations are consistent with the hypotheses that there is only one global species of giant squid, Architeuthis dux (Steenstrup, 1857), and that it is highly vagile, possibly dispersing through both a drifting paralarval stage and migration of larger individuals. Demographic history analyses of the genetic data suggest that there has been a recent population expansion or selective sweep, which may explain the low level of genetic diversity.

Brooding of egg masses by a squid in Japan is described. Brooding females were photographed in situ, and the females, their eggs, and their hatchlings were collected. The squid had all undergone gelatinous degeneration and swam slowly and continuously by undulating the fins and expelling water sporadically through the funnel. Eggs were held together by a dark, viscous material that formed a single-layer, sheet-like mass, from which hatchlings were seen to emerge. The annual appearance of brooding females in surface waters during spring suggests that they transport their egg masses from deep water to the surface before the eggs hatch. Genetic analyses identified the squid as Gonatus madokai (family Gonatidae), now the second gonatid and third squid known to brood.

Amyotrophic lateral sclerosis is a fatal neurodegenerative disease characterized by progressive motoneuron loss. Redistribution of transactive response deoxyribonucleic acid-binding protein 43 from the nucleus to the cytoplasm and the presence of cystatin C-positive Bunina bodies are considered pathological hallmarks of amyotrophic lateral sclerosis, but their significance has not been fully elucidated. Since all reported rodent transgenic models using wild-type transactive response deoxyribonucleic acid-binding protein 43 failed to recapitulate these features, we expected a species difference and aimed to make a non-human primate model of amyotrophic lateral sclerosis. We overexpressed wild-type human transactive response deoxyribonucleic acid-binding protein 43 in spinal cords of cynomolgus monkeys and rats by injecting adeno-associated virus vector into the cervical cord, and examined the phenotype using behavioural, electrophysiological, neuropathological and biochemical analyses. These monkeys developed progressive motor weakness and muscle atrophy with fasciculation in distal hand muscles first. They also showed regional cytoplasmic transactive response deoxyribonucleic acid-binding protein 43 mislocalization with loss of nuclear transactive response deoxyribonucleic acid-binding protein 43 staining in the lateral nuclear group of spinal cord innervating distal hand muscles and cystatin C-positive cytoplasmic aggregates, reminiscent of the spinal cord pathology of patients with amyotrophic lateral sclerosis. Transactive response deoxyribonucleic acid-binding protein 43 mislocalization was an early or presymptomatic event and was later associated with neuron loss. These findings suggest that the transactive response deoxyribonucleic acid-binding protein 43 mislocalization leads to α-motoneuron degeneration. Furthermore, truncation of transactive response deoxyribonucleic acid-binding protein 43 was not a prerequisite for motoneuronal degeneration, and phosphorylation of transactive response deoxyribonucleic acid-binding protein 43 occurred after degeneration had begun. In contrast, similarly prepared rat models expressed transactive response deoxyribonucleic acid-binding protein 43 only in the nucleus of motoneurons. There is thus a species difference in transactive response deoxyribonucleic acid-binding protein 43 pathology, and our monkey model recapitulates amyotrophic lateral sclerosis pathology to a greater extent than rodent models, providing a valuable tool for studying the pathogenesis of sporadic amyotrophic lateral sclerosis.

We have found a gene (thiP) encoding thiamine pyrophosphokinase (TPK) in the Aspergillus oryzae genome. No riboswitch-like region was found in the upstream region of thiP, although it was repressed probably by thiamine pyrophosphate (TPP) as well as thiA and nmtA, which are strictly regulated by TPP-riboswitch sequence. To improve the productivity of TPP in A. oryzae, we constructed the strain in which thiA, nmtA and thiP were overexpressed simultaneously. The resulting strain accumulated intracellular TPP 4-fold higher than did the control strain., (Copyright © 2010 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Systemic injections of AAV vectors generally transduce to the liver more effectively than to cardiac and skeletal muscles. The short hairpin RNA (shRNA)-expressing AAV9 (shRNA-AAV9) can also reduce target gene expression in the liver, but not enough in cardiac or skeletal muscles. Higher doses of shRNA-AAV9 required for inhibiting target genes in cardiac and skeletal muscles often results in shRNA-related toxicity including microRNA oversaturation that can induce fetal liver failure. In this study, we injected high-dose shRNA-AAV9 to neonates and efficiently silenced genes in cardiac and skeletal muscles without inducing liver toxicity. This is because AAV is most likely diluted or degraded in the liver than in cardiac or skeletal muscle during cell division after birth. We report that this systemically injected shRNA-AAV method does not induce any major side effects, such as liver dysfunction, and the dose of shRNA-AAV is sufficient for gene silencing in skeletal and cardiac muscle tissues. This novel method may be useful for generating gene knockdown in skeletal and cardiac mouse tissues, thus providing mouse models useful for analyzing diseases caused by loss-of-function of target genes., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Clinical information on familial amyotrophic lateral sclerosis (FALS) with Gly72Ser mutation in the Cu/Zn superoxide dismutase-1 (SOD1) gene has been limited and autopsy findings remain to be clarified. We describe one Japanese family with ALS carrying Gly72Ser mutation in the SOD1 gene, in which autopsy was performed on one affected member. The autopsied female patient developed muscle weakness of the left thigh at age 66 and showed transient upper motor neuron signs. She died of respiratory failure 13 months after onset without artificial respiratory support. There were no symptoms suggesting bladder or rectal dysfunction throughout the clinical course. Her brother with ALS was shown to have Gly72Ser mutation in the SOD1 gene. Histopathologically, motor neurons were markedly decreased throughout the whole spinal cord, whereas corticospinal tract involvement was very mild and was demonstrated only by CD68 immunohistochemistry. Degeneration was evident in the posterior funiculus, Clarke's nucleus, posterior cerebellar tract, and Onuf's nucleus. Neuronal hyaline inclusions were rarely observed in the neurons of the spinal cord anterior horn including Onuf's nucleus, and were immunoreactive for SOD1. To date, neuron loss in Onuf's nucleus has hardly been seen in ALS, except in the patients showing prolonged disease duration with artificial respiratory support. Involvement of Onuf's nucleus may be a characteristic pathological feature in FALS with Gly72Ser mutation in the SOD1 gene., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Gene therapy for dominantly inherited diseases with small interfering RNA (siRNA) requires mutant allele-specific suppression when genes in which mutation causes disease normally have an important role. We previously proposed a strategy for selective suppression of mutant alleles; both mutant and wild-type alleles are inhibited by most effective siRNA, and wild-type protein is restored using mRNA mutated to be resistant to the siRNA. Here, to prove the principle of this strategy in vivo, we applied it to our previously reported anti-copper/zinc superoxide dismutase (SOD1) short hairpin RNA (shRNA) transgenic (Tg) mice, in which the expression of the endogenous wild-type SOD1 gene was inhibited by more than 80%. These shRNA Tg mice showed hepatic lipid accumulation with mild liver dysfunction due to downregulation of endogenous wild-type SOD1. To rescue this side effect, we generated siRNA-resistant SOD1 Tg mice and crossed them with anti-SOD1 shRNA Tg mice, resulting in the disappearance of lipid accumulation in the liver. Furthermore, we also succeeded in mutant SOD1-specific gene suppression in the liver of SOD1(G93A) Tg mice, a model for amyotrophic lateral sclerosis, using intravenously administered viral vectors. Our method may prove useful for siRNA-based gene therapy for dominantly inherited diseases.

Twelve species of deep-sea fishes collected in 2005 from the western North Pacific, off-Tohoku, Japan were analyzed for organohalogen compounds. Among the compounds analyzed, concentrations of DDTs and PCBs (up to 23,000 and 12,400 ng/g lipid wt, respectively) were the highest. The present study is the foremost to report the occurrence of brominated flame retardants such as PBDEs and HBCDs in deep-sea organisms from the North Pacific region. Significant positive correlations found between delta(15)N ( per thousand) and PCBs, DDTs and PBDEs suggest the high biomagnification potential of these contaminants in food web. The large variation in delta(13)C (per thousand) values observed between the species indicate multiple sources of carbon in the food web and specific accumulation of hydrophobic organohalogen compounds in benthic dwelling carnivore species like snubnosed eel. The results obtained in this study highlight the usefulness of deep-sea fishes as sentinel species to monitor the deep-sea environment., (Copyright 2009 Elsevier Ltd. All rights reserved.)

In short hairpin RNA (shRNA) transgenic mice, the tissue difference in gene silencing efficiency and oversaturation of microRNA (miRNA) pathway have not been well assessed. We studied these problems in our previously-reported anti-copper/zinc superoxide dismutase (SOD1) shRNA transgenic mice. Although there was a tissue difference (liver and skeletal muscle, >95%; central nervous system and lung, approximately 80%), the target gene silencing was systemic and our anti-SOD1 shRNA transgenic mice recapitulated the SOD1-null mice. Neither endogenous miRNAs nor their target gene levels were altered, indicating the preservation of endogenous miRNA pathways. We think that the shRNA transgenic mice can be utilized for gene analysis.

Trace elements (22) and stable isotope ratios (delta15N and delta13C) were analyzed in marine organisms from shallow (SW) and deep-water (DW) of the East China Sea to understand biomagnification and prey source of trace elements. In the benthic marine organisms from DW, delta15N values were negatively correlated with Ba, Cu, Ag, Mo, Sr, As, and Co concentrations. This may be due to the specific accumulation in lower trophic animals and/or the biodilution through the food web in DW. Relationships between delta15N and concentrations of Co, Cr, Bi, and Tl in fish and Ag, Bi, V, Hg, and Tl in crustaceans showed positive correlations, suggesting that trophic position was affecting the concentrations of those elements in phyla, with higher trophic animals retaining higher concentrations than the lower trophic animals. Positive correlations between delta13C and Rb were observed in marine organisms. Therefore, Rb may be a possible substitute of delta13C as tracer of prey source in the East China Sea although further investigation is required.

RNA interference is a powerful tool for target-specific knockdown of gene expression. However, efficient and safe in vivo delivery of short interfering RNA (siRNA) to the target organ, which is essential for therapeutic applications, has not been established. In this study we used alpha-tocopherol (vitamin E), which has its own physiological transport pathway to most of the organs, as a carrier molecule of siRNA in vivo. The alpha-tocopherol was covalently bound to the antisense strand of 27/29-mer siRNA at the 5'-end (Toc-siRNA). The 27/29-mer Toc-siRNA was designed to be cleaved by Dicer, producing a mature form of 21/21-mer siRNA after releasing alpha-tocopherol. The C6 hydroxyl group of alpha-tocopherol, associated with antioxidant activity, was abolished. Using this new vector, intravenous injection of 2 mg/kg of Toc-siRNA, targeting apolipoprotein B (apoB), achieved efficient reduction of endogenous apoB messenger RNA (mRNA) in the liver. The downregulation of apoB mRNA was confirmed by the accumulation of lipid droplets in the liver as a phenotype. Neither induction of interferons (IFNs) nor other overt side effects were revealed by biochemical and pathological analyses. These findings indicate that Toc-siRNA is effective and safe for RNA interference-mediated gene silencing in vivo.

RNA interference (RNAi) represents a new technology which could offer potential applications for the therapeutics of human diseases. RNAi-mediated therapy has recently been shown to be effective toward infectious diseases in in vitro and rodent models, however, it remains unclear whether RNAi therapy with systemic application could be effective in primates. In this study, we examined if RNAi therapy could be effective toward infectious diseases by using a non-human primate surrogate model for hepatitis C. Administration into marmosets of cationic liposome-encapsulated siRNA (CL-siRNA) for GB virus B (GBV-B), which is most closely related to hepatitis C virus, repressed GBV-B replication in a dose-dependent manner. Especially, 5 mg/kg of the CL-siRNA completely inhibited the viral replication. Since the serum interferons (IFNs) were induced by CL-siRNA in vivo, inhibition of viral regulation by anti-GBV-B CL-siRNA may include an antiviral effect of IFN. However, contribution of induced IFN may be partial, since the control CL-siRNA which induced a stronger IFN response than GBV-B CL-siRNA could only delay the viral replication. Our results suggest the feasibility of systemic administration of CL-siRNA as an antiviral strategy.

Our newly developed underwater high definition video camera system took the first live images of adults of the mesopelagic large squid, Taningia danae, between 240 and 940 m deep off Ogasawara Islands, western North Pacific. The resulting footage includes attacking and bioluminescence behaviours, and reveals that T. danae is far from the sluggish neutrally buoyant deep-sea squid previously suspected. It can actively swim both forward and backward freely by flapping its large muscular triangular fins and changes direction quickly through bending its flexible body. It can attain speeds of 2-2.5 ms(-1) (7.2-9 km h(-1)) when attacking bait rigs. They emitted short bright light flashes from their large arm-tip photophores before final assault, which might act as a blinding flash for prey as well as a means of measuring target distance in a dark deep-sea environment. They also emitted long and short glows separated by intervals while wandering around the double torch lights attached to the bait rig, suggestive of potential courtship behaviours during mating.

The giant squid, Architeuthis, is renowned as the largest invertebrate in the world and has featured as an ominous sea monster in novels and movies. Considerable efforts to view this elusive creature in its deep-sea habitat have been singularly unsuccessful. Our digital camera and depth recorder system recently photographed an Architeuthis attacking bait at 900 m off Ogasawara Islands in the North Pacific. Here, we show the first wild images of a giant squid in its natural environment. Recovery of a severed tentacle confirmed both identification and scale of the squid (greater than 8 m). Architeuthis appears to be a much more active predator than previously suspected, using its elongate feeding tentacles to strike and tangle prey.

In gene therapy of dominantly inherited diseases with small interfering RNA (siRNA), mutant allele specific suppression may be necessary for diseases in which the defective gene normally has an important role. It is difficult, however, to design a mutant allele-specific siRNA for trinucleotide repeat diseases in which the difference of sequences is only repeat length. To overcome this problem, we use a new RNA interference (RNAi) strategy for selective suppression of mutant alleles. Both mutant and wild-type alleles are inhibited by the most effective siRNA, and wild-type protein is restored using the wild-type mRNA modified to be resistant to the siRNA. Here, we applied this method to spinocerebellar ataxia type 6 (SCA6). We discuss its feasibility and problems for future gene therapy.

We quantified the effect of Mg(2+) on thiamine pyrophosphate (TPP) binding to TPP-dependent thiA riboswitch RNA. The association constant of TPP binding to the riboswitch at 20 degrees C increased from 1.2 x 10(6) to 50 x 10(6) M(-1) as the Mg(2+) concentration increased from 0 to 1 mM. Furthermore, circular dichroic spectra under various conditions showed that 1 mM Mg(2+) induced a local structural change of the riboswitch, which might be pivotal for TPP binding. These results indicate that a physiological concentration of Mg(2+) can regulate TPP binding to the thiA riboswitch.

We report a case of relapsing polychondritis (RP) with an intracranial granuloma. A 67-year-old man developed progressive disorientation during the course of RP with left auricular chondritis and episcleritis. He had history of sinusitis and rupture of an aneurysm in middle cerebral artery. Laboratory examinations revealed high erythrocyte sedimentation rate and positive C-reactive protein. Head CT and MRI with contrast enhancement showed a mass adjacent to the falx cerebri and lesions in the frontal skull base. The mass was surrounded by extensive perifocal edema that spread mainly into the frontal white matter on both sides. Histologically, the mass displayed an inflammatory granuloma. By removal of the mass, edema decreased around the granuloma, and his disorientation improved markedly. Surgical findings revealed the granuloma was separated from sinusitis. There are a few reports on RP with an intracranial granuloma.

Exogenous thiamine regulates Aspergillus oryzae thiA, which is involved in thiamine synthesis. One of the two introns in its 5'-untranslated region (5'-UTR) contains motifs (regions A and B) highly conserved among fungal thiamine biosynthesis genes. Deletion of either region relieved the repression by thiamine and thiamine inhibited intron splicing, suggesting that regions A and B are required for efficient splicing. Furthermore, transcript splicing was essential for thiA gene expression. These observations suggest a novel gene expression regulatory mechanism in filamentous fungi, in which exogenous thiamine controls intron splicing to regulate gene expression. Interestingly, regions A and B constitute a part of a thiamine pyrophosphate-binding riboswitch-like domain that has been quite recently found in the 5'-UTR of thiA.

Spinocerebellar ataxia type 6 (SCA6) is a neurodegenerative disease caused by small CAG repeat expansion in the alpha1A calcium channel gene. We found that the human alpha1A calcium channel protein expressed in human embryonic kidney 293T cells produces a 75 kDa C-terminal fragment. This fragment is more toxic to cells than the full-length alpha1A calcium channel, regardless of polyglutamine tract length. In cells stably transfected with plasmids of full-length alpha1A calcium channel cDNAs, the C-terminal fragment protein is present in the mutant transformant but not in the wild-type one, indicative that this C-terminal fragment with the expanded polyglutamine tract is more resistant to proteolysis than that with the normal sized polyglutamine tract. We speculate that the toxic C-terminal fragment, in which resistance to proteolysis is rendered by the expanded polyglutamine, has a key role in the pathological mechanism of SCA6.

Mureka-non-forming sake koji molds were constructed from an Aspergillus oryzae industrial strain by the disruption of the mreA gene using a host-vector system with the ptrA gene as a dominant selectable marker. All of the mreA gene disruptants obtained retained the advantages of the host strain in terms of the brewing characteristics, while their isoamyl alcohol oxidase (IAAOD) activities were significantly lower than that of the host strain. Sake brewing was successfully carried out using the koji prepared with the disruptants, followed by storage of the resultant non-pasteurized sake (nama-shu). The isovaleraldehyde (i-Val) concentration in the sake brewed the host strain increased rapidly and reached the threshold values for mureka, 1.8 ppm and 2.6 ppm after storage at 20 degrees C for 42 d and 63 d, respectively, while those of the disruptants were less than 0.5 ppm even after storage at 20 degrees C or 30 degrees C for 63 d. In the sensory evaluation of the sake stored at 20 degrees C or 30 degrees C for 63 d, all members of the panel recognized the strong mureka flavor of the sake brewed with the host strain, while they did not detect this flavor in the sake brewed with the disruptants. Thus, we concluded that the mreA gene disruptants can be used for the production of sake in which mureka is not formed.

A pyrithiamine (PT) resistance gene (ptrA) was cloned from a PT resistant mutant of Aspergillus oryzae and was useful as a dominant selectable marker for transformation of all A. oryzae wild type strain as well as A. nidulans. For further study, we examined whether or not ptrA could be used as the transformation marker in other species of filamentous fungi. Two types of plasmid, which contain ptrA as a selectable marker, were constructed, and the transformation experiments were done with them. One is an integrative plasmid, pPTRI, and another is the autonomously replicating plasmid pPTRII, which contains AMA1. PT-resistant transformants were obtained in the cases of A. kawachii, A. terreus, A. fumigatus, and Trichoderma reesei as hosts with pPTRI and pPTRII. Furthermore, a beta-glucuronidase (GUS) gene was introduced into A. kawachii and A. fumigatus using pPTRII. Almost all the transformants turned blue on GUS assay plates. These results indicate that ptrA can also be used for some other filamentous fungi besides A. oryzae and A. nidulans.

Pure autonomic failure (PAF) is an uncommon disorder, characterized by autonomic failure without other neurological deficits. We report here an autopsy case of pure autonomic failure with pathological features of Parkinson's disease. At age 79, the patient developed slowly progressive severe orthostatic hypotension, followed by decreased sweating and constipation. On admission, his blood pressure dropped after standing (from 133/97 to 71/22 mmHg) without tachycardia. Neurological examination revealed neither parkinsonism, nor cerebellar symptoms. Supine plasma norepinephrine levels were low and intravenous norepinephrine administration showed denervation hypersensitivity. He was diagnosed as having PAF and treated by amezinium, midodrine, L-threodops, and fludrocortisone with elastic stockings but without any effects. He died of pneumonia about 2 years after the onset of the disease. Postmortem study revealed Lewy bodies in the nucleus basalis of Meynert, substantia nigra. Edinger-Westphal nucleus, locus ceruleus, and dorsal vagal nucleus. Neuronal loss was found in above-mentioned sites, except for Edinger-Westphal nucleus and intermediolateral nucleus. This is a rare and important report on a patient with PAF who satisfied the strict clinical criteria on PAF reported in 1996, and showed neuropathological findings, consistent with Parkinson's disease.

A pyrithiamine (PT) resistance gene (ptrA) was cloned from a genomic DNA library prepared from a PT resistant mutant of Aspergillus oryzae. It conferred high resistance to PT on an A. oryzae industrial strain as well as A. nidulans. Nucleotide sequence analysis showed that the ptrA gene contained one intron (58-bp) and encodes 327 amino acid (aa) residues. Additionally, the deduced aa sequence has 72% and 63% identity to Fusarium solani sti35 encoding a stress-inducible protein and Saccharomyces cerevisiae THI4 encoding an enzyme involved in thiamine biosynthesis, respectively, indicating that ptrA is a mutated allele of a gene belonging to the THI4 family. The mutation point was identified in the conserved motif in 5'-flanking region of these three THI4 homologous genes (ptrA, sti35, and THI4). The introduction of the ptrA gene allowed an A. oryzae industrial strain to grow on the minimum medium containing PT (0.1 mg/l) on which an untransformed strain did not grow. This result indicates that the ptrA is applicable as a dominant selectable marker for transformation of A. oryzae.

The cultivable subgingival bacterial flora from three HIV-seropositive and CD4 cell depleted children with hemophilia were examined in this study. The numbers of CD4 cells of the subjected children ranged from 4.9 to 16.3 per mm3 blood. Streptococcus species, including, beta-hemolytic streptococcus identified as Streptococcus mutans and Streptococcus sanguis, were predominant in the subgingival plaque samples. Actinomyces species were also frequently found. Gram-negative rods other than Capnocytophaga species were not common in these samples. It is possible that the subgingival microbial flora are influenced by the CD4 cell decrease with HIV infection.

The cortex and medulla areas, sectioned frontally, of 50 kidneys from 25 dissecting cadavers were measured to determine the correlation between differences in sex, laterality, and age. The average parenchyma (cortex + medulla) area was 33.45 +/- 9.16 cm2 and was larger in men than women; there was no significant laterality, but the parenchyma area was negatively correlated with age. The average cortex area was 20.39 +/- 5.80 cm2 and was also larger in men than women; laterality was not significant, and the cortex area was negatively correlated with age. The average medulla area was 13.05 +/- 4.40 cm2, and no significant difference in sex or laterality was found. The medulla area was negatively correlated with age. Additionally, the average number of renal pyramids (medulla) was 6.24 +/- 1.45 on these sections. Difference in the average areas of cortex and medulla was significant. The medulla was exceptionally larger in two cases (4%). The ratio between the average area of the cortex and medulla (C/M ratio) was 1.67 +/- 0.56. No sex difference or laterality was found, nor was there a correlation between the C/M ratio and age or kidney size. The sectioned area of cortex was approximately 1.5 to 2 times larger than medulla. These results suggest that senile atrophy may occur at the same rate in the renal cortex as in the medulla.

An enzyme producing isoprimeverose from xyloglucan fragment oligosaccharides has been purified to the electrophoretically pure state from a commercial enzyme preparation of Aspergillus oryzae (Sanzyme 1000). The purified enzyme showed approximately 1,280-fold increase of the specific activity over the original preparation. The purified enzyme was shown to be an oligomeric protein consisting of two subunits, each of which had a molecular weight of 115,000. The enzyme showed the highest activity at pH 5.0 and 60 degrees C, and was stable in the pH range from 5 to 7 and at up to 50 degrees C. The isoelectric point of this enzyme was pH 3.9. The purified enzyme was highly specific for xyloglucan fragment oligosaccharides and split off isoprimeverose units from the non-reducing end of the backbone of the substrate.

Five cases of the double renal pelvis and ureter were encountered among 340 cadavers for dissection practice. Number of minor calices and their surface area in these kidneys were calculated, and causal relationship to abnormal organogenesis was discussed. These 5 cases were formal exclusively in consist of males; 4 right sides and 1 left side, 2 complete duplications and 3 partial duplications. The 5 cases were divided into 2 groups by the number and surface area of minor calices in upper and lower pelvises as follows: 1) The number and surface area of minor calices in the upper pelvis and the lower pelvis were respectively equivalent to the mean value of those in one normal kidney (case 1). 2) The sum of the number and surface area of minor calices in the upper and those in the lower pelvises were equivalent to the mean value of those in one normal kidney (case 2-5). These cases give useful criteria for classification of the double renal pelvis and ureter.

A new compact high-performance instrument for monitoring the oxygenation state of the human newborn are developed. First we applied this instrument to the rat head, and next to the human arm. We could confirm that the volume of Oxy-Hb, Deoxy-Hb and total blood volume were well monitored by using our instrument. Now we started to apply this technique to the human newborn. Furthermore, we started the experiments of the near-infrared tomography for the small animals on the basis of this technique.

A fundamental study was performed to establish a method of quantitative analysis using mass chromatography by the direct inlet system. The samples were aspirin, phenacetin and caffeine, which are often used as cold medicines, and barbital, allobarbital, phenobarbital and phenytoin which are difficult to analyse by gas chromatography in their intact states. N-acetylsulfamine and ethyl p-aminobenzoate were used as internal standards. Direct inlet mass chromatography was performed by an on-line system of the Shimadzu LKB-9000 and the GC-MSPAC 300. The ratio of the cumulative ions of certain peaks of sample and the internal standard was studied. It was found that, whether the sample is a pure reagent or a mixture, the ratio of cumulative ions of a peak specific to the sample and of a selected peak of the internal standard is proportional to the sample size, the error being less than +/- 3.5% for aspirin, phenacetin and caffeine, and less than +/- 2.7% for barbital, allobarbital and phenobarbital. The same relationship was observed for the phenobarbital and phenytoin mixed in rat plasma, the error being less than +/- 2.0%. It can be concluded that this method is applicable to the quantiative determination of medicines in urine, body fluids and other biological samples.