Poly(adenosine diphosphate-ribose) synthetase inhibitor 3-aminobenzamide alleviates cochlear dysfunction induced by transient ischemia.

The present study was undertaken to determine the possible deleterious role played by poly(adenosine diphosphate-ribose) synthetase (PARS) in cochlear ischemia-reperfusion injury. Transient ischemia of the cochlea was induced in albino guinea pigs for 15, 30, or 60 minutes by pressing the labyrinthine artery at the porus acusticus internus. The animals were given intravenous 3-aminobenzamide (a PARS inhibitor) or physiological saline solution I minute before the onset of reperfusion. The compound action potential thresholds were measured before the onset of ischemia and 4 hours after the onset of reperfusion. A statistically significant reduction in the postischemic compound action potential threshold shift was observed in the animals treated with 3-aminobenzamide after 15 or 30 minutes of ischemia, whereas no statistical difference was found after 60 minutes of ischemia. These results suggest that excessive activation of PARS exerts deleterious effects on the cochlear injury induced by transient ischemia.

Identification and characterization of newt rad (ras associated with diabetes), a gene specifically expressed in regenerating limb muscle.

Formation of the blastema is a key event for limb regeneration in urodele amphibians, and skeletal muscle has been thought to be a major origin of the multipotent blastemal mesenchyme. In the present study, we used differential display to identify the genes expressed differentially in the muscle at the amputation site. We have isolated a cDNA clone that was upregulated during limb regeneration of the Japanese newt, Cynops pyrrhogaster. Deduced amino acid sequence revealed that the cloned cDNA was a newt homolog of rad (ras associated with diabetes), a gene overexpressed in skeletal muscle of Type II diabetic patients. Expression of newt rad (nrad) was not observed in unamputated normal limb muscle, increased within 4 hr after amputation, and then decreased to the level of normal muscle between 11 and 21 days after amputation. In situ hybridization showed that the transcripts of nrad were localized around most of the nuclei of skeletal muscle near the amputation site, indicating the expression of nrad in the multinucleate myotubes. This expression gradually decreased along the distal to proximal axis. No signals were observed in apical epidermal cap or blastemal mesenchyme. However, reverse transcription-PCR analysis detected a very low level of nrad expression in blastema, suggesting the carry-over of nrad expression in blastema from muscle. Administration of retinoic acid, which has been shown to cause an enhanced dedifferentiation in the regenerating limbs, increased nrad expression in more proximally located limb muscle tissues and prolonged the expression period. Thus, it was strongly suggested that the nrad expression is correlated with the dedifferentiation of myotubes of regenerating limbs. We also analyzed the expression of nrad during development. Transcripts were observed in immature oocytes, seen faintly or not seen thereafter until stage 57 when its expression increased again. These results indicated that nrad may play a role(s) in the developmental process as well as limb regeneration.

[Treatment of status epilepticus].

Status epilepticus (SE) is a condition requiring emergency care. There are convulsive SE, non-convulsive SE including complex partial status and absence status, non-convulsive electric SE and pseudostatus epilepticus, although convulsive SE is the most common. Diagnosis of status epilepticus of complex partial seizures (CPS) and absence seizures was significantly delayed because delays in seeking medical attention were common. The seizures were generalized convulsive SE in 84% and CPS status in 16%, and the overall mortality rate was 15% in 41 SE patients of our study. EEG monitoring is important to make or exclude the diagnosis of SE. Diazepam is the first choice medication and effective in the management of SE, and lately, lorazepam, midazolam, propofol and pentobarbital etc as emergency therapy. Phenytoin is also considered first-line agent in the emergency management of SE. Repetitive transcranial magnetic stimulation (rTMS) led to a prolonged latency for seizure induction after an intraperitoneal injection of pentylenetetrazol (PTZ) and effectively prevented the development of status epilepticus of PTZ-induced convulsions in the rats. Our data suggest that rTMS has suppressive effects on the neuronal excitability in rats. These effects are anticonvulsive and suggest the possibility of therapeutic use of rTMS in the patients with refractory seizures.

[An early-onset case of acute disseminated encephalomyelitis with bilateral thalamic lesions on MRI].

We report the case of 5-year-old girl with acute disseminated encephalomyelitis (ADEM), whose MRI showed bilateral thalamic lesions. She suffered from left optic neuritis and generalized convulsion. Examination of cerebrospinal fluid revealed elevation of mononuclear cells and myelin basic protein (MBP). MRI showed the swelling of left optic nerve and high intensity areas of bilateral thalamus. After methylprednisolone pulse therapy, her visual acuity was dramatically improved and bilateral thalamic lesions were decreased. In childhood, bilateral thalamic lesions were observed in several diseases, such as viral encephalitis. Reye syndrome, Leigh syndrome and acute necrotizing encephalopathy. Demyelinating diseases involving the grey matter were very rare, but we must consider the presence of symmetrical thalamic involvement in patients with ADEM.

Isolation of 10 differentially expressed cDNAs in differentiated Neuro2a cells induced through controlled expression of the GD3 synthase gene.

Recently, we showed that transfection of GD3 synthase cDNA into Neuro2a cells, a mouse neuroblastoma cell line, causes cell differentiation with neurite sprouting. In a search for the genes involved in this ganglioside-induced Neuro2a differentiation, we used a tetracycline-regulated GD3 synthase cDNA expression system combined with differential display PCRs to identify mRNAs that were differentially expressed at four representative time points during the process. We report here the identification of 10 mRNAs that are expressed highly at the Neuro2a differentiated stage. These cDNAs were named GDAP1-GDAP10 for (ganglioside-induced differentiation-associated protein) cDNAs. It is interesting that in retinoic acid-induced neural differentiated mouse embryonic carcinoma P19 cells, GDAP mRNA expression levels were also up-regulated (except that of GDAP3), ranging from three to >10 times compared with nondifferentiated P19 cells. All the GDAP genes (except that of GDAP3) were developmentally regulated. The GDAP1, 2, 6, 8, and 10 mRNAs were expressed highly in the adult mouse brain, whereas all the other GDAP mRNAs were expressed in most tissues. Our results suggested that these GDAP genes might be involved in the signal transduction pathway that is triggered through the expression of a single sialyltransferase gene to induce neurite-like differentiation of Neuro2a cells.

Pure apraxic agraphia with abnormal writing stroke sequences: report of a Japanese patient with a left superior parietal haemorrhage.

A 67 year old Japanese male patient had pure agraphia after a haemorrhage in the left superior parietal lobule. He developed difficulty in letter formation but showed no linguistic errors, consistent with the criteria of apraxic agraphia. He manifested a selective disorder of sequencing writing strokes, although he was able to orally state the correct sequences. The patient's complete recovery after 1 month, without new learning, showed that he had manifested a selective disorder of writing stroke sequences. These findings indicate that the final stage of the execution of writing according to acquired sequential memory shown as a stroke sequence can be selectively disturbed, and should be considered to be distinct from the ability of character imagery and the knowledge of the writing stroke sequence itself. This case also indicates that the left superior parietal lobule plays an important part in the execution of writing.

BASH, a novel signaling molecule preferentially expressed in B cells of the bursa of Fabricius.

The bursa of Fabricius is a gut-associated lymphoid organ that is essential for the generation of a diversified B cell repertoire in the chicken. We describe here a novel gene preferentially expressed in bursal B cells. The gene encodes an 85-kDa protein, designated BASH (B cell adaptor containing SH2 domain), that contains N-terminal acidic domains with SH2 domain-binding phosphotyrosine-based motifs, a proline-rich domain, and a C-terminal SH2 domain. BASH shows a substantial sequence similarity to SLP-76, an adaptor protein functioning in TCR-signal transduction. BASH becomes tyrosine-phosphorylated with the B cell Ag receptor (BCR) cross-link or by coexpression with Syk and Lyn and associates with signaling molecules including Syk and a putative chicken Shc homologue. Overexpression of BASH results in suppression of the NF-AT activation induced by BCR-cross-linking. These findings suggest that BASH is involved in BCR-mediated signal transduction and could play a critical role in B cell development in the bursa.

Immunopotentiating activities of polysaccharide fraction from pine seed shell extract.

The function of polysaccharide (PSE) extracted from pine seed shells as a immunopotentiator was investigated. The phagocytosis and chemotaxis of mouse peritoneal macrophages (MP) were augmented by oral administration of PSE. The incorporation of 3H-thymidine by Con A-stimulated mouse splenic cells was significantly intensified by administration of PSE but the adherent-cells (MP)-eliminated splenic lymphocytes was not increased. It is shown that the existence of activated MP is needed for the activation (proliferation) of T lymphocytes. The responsiveness of mouse T cells to alloantigen was also augmented by administration of PSE. The number of anti-SRBC plaque-forming cells in the spleen of mouse and chicken was also significantly increased. The results indicate that MP are first activated by oral administration of PSE and that the activation of T cells is then initiated indirectly by humoral factors (cytokines) produced by activated MP.

Thalamic form of Creutzfeldt-Jakob disease or fatal insomnia? Report of a sporadic case with normal prion protein genotype.

We describe a 68-year-old man with a 53-month history of progressive dementia and clinical features of a progressive supranuclear palsy-like syndrome and dysautonomia. In the late stage of his illness, the patient also developed generalized myoclonic seizures. There was no family history of similar disorders. Histological examination revealed neuronal loss and gliosis with spongiosis in the cerebral cortex. In addition, more severe neuronal loss and gliosis without spongiosis were observed in the thalamus, especially in the anterior ventral and mediodorsal nuclei, and the inferior olivary nucleus. There was also obvious loss of Purkinje cells. Immunohistochemically, no protease-resistant prion protein (PrPres)-positive structures were demonstrated. However, Western blotting revealed the presence of PrPres in the cerebral cortex. This patient had a wild type of PrP genotype. We initially considered this to be a case of the thalamic form of Creutzfeldt-Jakob disease (CJD) with a long duration. However, it is noteworthy that essentially similar pathology, albeit with less severe cerebral cortical changes, has also been reported in fatal familial insomnia, a newly identified phenotypically different prion disease with a mutation in the PrP gene. On the basis of clinicopathological features, we eventually felt that this patient was more likely to have been a sporadic case of fatal insomnia (FI) of long duration. The present case appears to draw further attention to the possible relationship between CJD and FI.

Two polysialic acid synthases, mouse ST8Sia II and IV, synthesize different degrees of polysialic acids on different substrate glycoproteins in mouse neuroblastoma Neuro2a cells.

We previously cloned cDNAs encoding two different polysialic acid (PSA) synthases, ST8Sia II and IV, from mouse, and showed that both mouse ST8Sia II and IV can synthesize PSA on the neural cell adhesion molecule (NCAM) as well as other glycoproteins such as fetuin, at least in vitro (Kojima, N., Tachida, Y., Yoshida, Y., and Tsuji, S. (1996) J. Biol. Chem. 271, 19457-19463]. In the present study, to clarify how the two PSA synthases act differently in vivo, we first cloned PSA-expressing cell lines (N2a-II and N2a-IV) by stable transfection of the cDNA encoding either mST8Sia II or IV into mouse neuroblastoma Neuro2a cells, which do not express PSA but express NCAM, then compared the expression of the PSA and NCAM isoforms and de novo synthesis of PSA between N2a-II and N2a-IV. Western blotting with an anti-NCAM polyclonal antibody showed that NCAM was expressed as the polysialylated form in both ST8Sia II cDNA-transfected and ST8Sia IV cDNA-transfected Neuro2a cells, but that the polysialylated NCAMs expressed in ST8Sia IV cDNA-transfected clones migrated much slower on SDS-PAGE than those expressed in ST8Sia II cDNA-transfected clones. The slower migration of polysialylated NCAM of the ST8Sia IV cDNA-transfected clone (N2a-IV) than that of the ST8Sia II cDNA-transfected clone (N2a-II) was also observed when cells were metabolically labeled with [3H]glucosamine or pulse-chase labeled with [35S] methionine followed by immunoprecipitation with anti-PSA antibody or anti-NCAM monoclonal antibody. In addition, polysialylated N-glycans of PSA-carrying glycoproteins prepared from [3H] glucosamine-labeled N2a-IV by immunoprecipitation with anti-PSA monoclonal antibody were eluted at a much higher salt concentration than those from [3H] glucosamine-labeled N2a-II on an anion-exchange column. These results indicated that the degree of de novo polysialylation of NCAM by mST8Sia IV was much higher than that by mST8Sia II. In N2a-IV, NCAM-120, -140, and -180 were expressed as polysialylated forms, while polysialylation was restricted to NCAM-140 and -180, i.e., not NCAM-120, in N2a-ST8Sia II. Metabolic labeling of the cells with [3H] glucosamine, pulse-chase labeling with [35S] methionine followed by immunoprecipitation with anti-PSA antibody, and subsequent sialidase treatment revealed that NCAM-140 and -180 were specifically polysialylated in N2a-II, whereas not only NCAM but also other glycoproteins were de novo polysialylated in N2a-IV. The above results demonstrated that the two different PSA synthases, mST8Sia II and IV, synthesize PSA of different lengths on different substrate glycoproteins in vivo when the enzymes are expressed in neuroblastoma Neuro2a cells. These differences suggest that mST8Sia II and IV play different roles in the biosynthesis and expression of PSA.

Simultaneous observation of regional cerebral blood flow and event-related potential during performance of an auditory task.

Reduced amplitude of the P300 component has been reported consistently in patients with neurological and psychiatric disorders. It is unclear, however, how such patients' cognitive dysfunction is related to their P300 abnormality. Further basic knowledge regarding neural substrates for P300 generation is required for gaining an understanding of the pathological significance of the P300 amplitude reduction. To determine the brain structures involved in P300 generation, we observed the event-related potential and the regional cerebral blood flow (rCBF) in 10 normal subjects performing an oddball discrimination of pure tones. The rCBF value was assessed quantitatively with the aid of single photon emission computed tomography using technetium-99m hexamethylpropylene amine oxime. During the task performance, significant activation was observed in the posterior superior temporal and inferior parietal regions of the right hemisphere. In addition, positive correlation of the task-related increase in rCBF with the simultaneously recorded P300 amplitude was observed in the right but not the left posterior superior temporal region. These findings indicate that activation of the right non-verbal auditory area might modulate P300 generation during pure-tone discrimination.

Instability of mutant Cu/Zn superoxide dismutase (Ala4Thr) associated with familial amyotrophic lateral sclerosis.

In about 20-25% of cases of familial amyotrophic lateral sclerosis (FALS) patients have mutations in the Cu/Zn superoxide dismutase (SOD1) gene. The mechanism through which the mutations in the SOD1 gene cause ALS still remain unknown. We performed pulse-chase experiments using a system for the transient expression of human SOD1 in COS7 cells to examine whether the Ala4Thr mutation, which we previously reported, decreases the stability of SOD1. The expression vector (pEF-BOS) carrying the wild-type or mutant (Ala4Thr) human SOD1 cDNA was transfected into COS7 cells, and transiently expressed human SOD1 was then metabolically radiolabeled. Half-lives of the wild-type and the Ala4Thr mutant SOD1 were determined to be 78 h and 18 h, respectively. These results suggest that the Ala4Thr mutation in SOD1 decreases the stability of SOD1 and that this instability may play an important role in the pathogenesis of the degeneration of motor neurons in FALS.

Ipsilateral hemiplegia caused by right internal capsule and thalamic hemorrhage: demonstration of predominant ipsilateral innervation of motor and sensory systems by MRI, MEP, and SEP.

A patient with a right internal capsule and thalamic hemorrhage showed ipsilateral hemiplegia. MRI at 10 months after the cerebral hemorrhage demonstrated Wallerian degeneration, which could be traced to the ipsilateral anterior funiculus at the cervical level. The findings of motor evoked potentials and somatosensory evoked potentials indicate a predominantly ipsilateral innervation of motor and sensory systems in this particular patient.

[Relation between defect in comprehension and frontal lobe lesion].

We assessed anatomical findings and language defects in 14 right handed patients who had fluent aphasia following left frontal lobe lesion. From the onset of aphasia all of the patients showed fluent speech and excellent repetition but difficulty in word finding and impairment in language comprehension. We administered to all of the patients the Western Aphasia Battery, a 50-item pointing task using line drawings representing single words selected from among common Japanese words for language training for aphasics, and the Token Test. Anatomical analysis was performed using brain CT and/or MRI. The patients were divided into three groups on the basis of the extent of impairment in comprehension of single words: one group showed no impairment, another showed slight impairment and the other showed severe impairment. The lesion site differed among the groups. Each group had a different lesion site. We concluded the following: first, lesions in Brodmann's areas 6 and 9 produce impairment in comprehension of single words, with lesions extending to anterior to Broca's area producing more impairment than those without the extending lesions. Second, lesions in the frontal lobe produce impairment in comprehension of complex sentences.

Isolation of human purH gene expressed in the rodent transformant cells by subtractive enrichment of 3'-untranslated region of human transcript.

A subtraction procedure was developed for identification and isolation of a human gene transcribed in mouse transformant cells. The procedure was based on subtractive enrichment of the products that were amplified by the combination of reverse transcription and polymerase chain reaction from the 3'-untranslated region (3'-UTR) of human poly(A)+ RNA expressed in the mouse transformant cells. To assess the ability and usefulness of the procedure, we attempted to recover the human purH gene from a mouse transformant cell line, which was originally established by functional complementation using the human metaphase chromosome-mediated gene transfer technique from a mouse purH-negative mutant cell line. Using our procedure, a part of the human transcript in the transformant cells was successfully identified and isolated. The full-length cDNA was isolated using the 3'-UTR clone as a probe, and its biological activity was confirmed by introducing it into the mouse purH-negative mutant cells.

[Intratumoral LPD deposition within hepatocellular carcinoma without arterial tumor staining].

The dissociation of intratumoral LPD deposition and arterial tumor staining was investigated in 20 cases of hepatocellular carcinoma (HCC) without arterial tumor staining. The intratumoral deposition of LPD injected from the hepatic artery was observed in nine cases of HCC and coincided with the decrease in tumor vascular bed evaluated by photomicroscopic study and arterial portography. LPD deposition was not observed within the 11 other tumors, which were diagnosed as well differentiated HCC. Infusion angiographic CT revealed that the arterial and portal vascular beds were separated from each other. Therefore, arterial tumor staining was insufficient for accurate evaluation of the tumor vascular bed. The vascular bed in cancerous and noncancerous tissue was accurately evaluated by the addition of the contrast enhancements from the hepatic artery and the portal vein.

Comparative SPECT study of stroke using Tc-99m ECD, I-123 IMP, and Tc-99m HMPAO.

Brain perfusion SPECT imaging using Tc-99m ethyl cysteinate dimer (ECD) was carried out in 12 patients with chronic cerebrovascular disease. The sensitivity of lesion detection and lesion-to-normal tissue contrast was compared with those of I-123 IMP and Tc-99m HMPAO. Eight patients underwent all three studies and the remaining four patients had Tc-99m ECD and I-123 IMP scans. The sensitivity of lesion detection and lesion contrast was analyzed quantitatively using an asymmetric index determined by the equation: 100 x [right - left[/(right + left). In the cerebral cortex, cerebellum, and white matter, I-123 IMP showed higher lesion sensitivity than Tc-99m ECD and Tc-99m HMPAO. In the striatum and thalamus, Tc-99m ECD and I-123 IMP showed higher lesion sensitivity than Tc-99m HMPAO. I-123 IMP showed the highest lesion contrast in cerebral cortex and cerebellum, whereas Tc-99m ECD showed the highest contrast in thalamus and striatum. In all regions, Tc-99m ECD showed higher lesion contrast than Tc-99m HMPAO. These results suggest regional variation in the sensitivity of lesion detection and lesion contrast of Tc-99m ECD compared to I-123 IMP, and the superiority of Tc-99m ECD to Tc-99m HMPAO.

Biodistribution of intravenously injected [131I]Lipiodol in rats.

Tissue distribution studies of intravenously injected [131I]Lipiodol in rats were performed, and serial whole-body autoradiograms were obtained simultaneously for seven days. Most of the radiotracer was retained in the lungs. Lung uptake reached a maximum (42.44% ID/g) at 1 day, with an effective half-life of 3.0 days. The other organs showed markedly lower uptakes (less than 0.56% ID/g). The ratio between liver and lung reached a maximum (0.01) at 3 hr and then decreased with time, precluding adequate external imaging of the liver as compared with the lung.

Comparison of lactoferrin content in colostrum between different cattle breeds.

Lactoferrin content of colostrum obtained from cows within 24 h after parturition was measured using a single radial immunodiffusion test and was compared among cows of two dairy breeds (Holstein-Friesian, Jersey) and two beef breeds (Japanese Black and Japanese Brown). Average lactoferrin content in colostrum of dairy breeds was 2 mg/ml and in colostrum of beef breeds was .5 mg/ml. Lactoferrin content of colostrum due to lactation number was also different among breeds. In dairy breeds, multiparous cows had lactoferrin content two to three times higher than that of primiparous cows; beef breeds showed no obvious differences between lactation years. Lactoferrin content also varied considerably within breed. In beef breeds, half the cows had values of nearly zero. Transferrin content in colostrum was fairly constant (.9 mg/ml) and was not as variable among and within breeds. There was no correlation between lactoferrin and transferrin contents in colostrum. Examination of cows lacking lactoferrin suggested that transferrin plays an important role as an iron carrier from a cow to her newborn calf.