Kiyofumi Saijoh

Molecular cloning of cDNA encoding a bovine selenoprotein P-like protein containing 12 selenocysteines and a (His-Pro) rich domain insertion, and its regional expression.

When cDNA containing proteins enriched in the bovine cerebellar cortex were cloned, a clone which seemed to encode a selenoprotein P-like protein was isolated. The coding nucleotide sequence of its cDNA insert displayed high homology to rat and human selenoprotein P cDNA but contained 12 rather than 10 TGAs (12 rather than 10 selenocysteines in deduced amino acids), a tandem repeat of one CACTCC (His-Ser) and seven CATCCCs (His-Pro), and a 3 untranslated region approximately 890 bases shorter than that of rat liver selenoprotein P. RT-PCR using a set of primers flanking to the repeat displayed the existence of mRNA without the repeat. The tandem repeat and its adjacent region consisted of a similar motif of CAC/TCC/AC/T. Thus, these proteins included a (His-Pro) rich domain with a slightly negative free energy change irrespective of having the tandem repeat or not. Such His-Pro repeats reportedly exist in the segmentation gene paired or homeobox protein Om(1D) of Drosophila. Moreover, both this selenoprotein P-like protein mRNA and selenoprotein P mRNA were expressed in all the areas of the brain but most prominently in the cerebellar cortex, hippocampus, and olfactory bulb. These findings suggest the possibility that these selenoproteins are major selenium carriers in the brain and play a role in the morphological response of nerve or glial cells.

Solubilization of D2 Dopamine Receptor Coupled to Guanine Nucleotide Regulatory Protein from Bovine Striatum

Abstract: D2 dopamine receptor from bovine striatum was solubilized in a form sensitive to guanine nucleotides, by means of a zwitterionic detergent, 3–[(3‐cholamidopropyl)‐dimethylammonio]‐1 ‐propane sulfonate (CHAPS). The presence of sodium ion markedly increased the solubilization yield. Treatment of the membranes with 10 mM CHAPS and 0.72 M NaCl solubilized 26% of the stereospecific [3H]spiperone binding sites in the original membrane preparations. The solubilized [3H]spiperone binding sites possessed characteristics of the D2 dopamine receptor: (a) localization of the site in the striatum but not in the cerebellum; (b) high affinity to nanomolar concentrations of [3H]spiperone; (c) displacement of [3H]‐spiperone binding by nanomolar concentrations of neuroleptics, but only by micromolar concentrations of do pamine and apomorphine; (d) equal activity of various dopamine agonists and antagonists in the soluble and membrane preparations. Guanine nucleotides decreased the affinity of the solubilized D2 dopamine receptor for dopamine agonists, but not for antagonists. The solubilized receptor complex was eluted in Sepharose CL‐4B column chromajography as a large molecule, with a Stokes radius of ∼90 Å. These results indicate that the complex between the D2 dopamine receptor and GTP binding protein remains intact throughout the solubilization procedure.

Human Papillomavirus Infection and Risk Determinants for Squamous Intraepithelial Lesion and Cervical Cancer in Japan

A case control design was used to investigate human papillomavirus (HPV) prevalence and risk factors associated with development of cervical squamous intraepithelial lesion (SIL) and cervical cancer (CC) in Japan, One hundred and twenty‐three women with histologically confirmed SIL or CC were compared to a control group of 778 cytologically normal women. With the use of a polymerase chain reaction (FCR)‐hased method for detection of low‐risk (types 6 and 11) and high‐risk (types 16, 18, 31, 33, 35, 52 and 58) HPVs, a high prevalence of HPV infection was observed in smokers among the controls. Logistic regression analysis demonstrated that high‐risk HPV infection was the most significant risk determinant for LSIL (OR=9.4, 95% CI=4.5–19), HSIL (OR=77, 95% CI=28–217) and CC (OR=97, 95% CI=35–269). It also showed that unmarried women, women married for 5 to 19 years and smokers represented high risk groups for SIL, while smokers and women with a history of many pregnancies/parities had increased risk for CC. Smoking was the only HPV infection‐independent factor for CC, suggesting that smoking may have a carcinogenic effect on the cervix. Since neither history of other cancer nor family cancer history was associated with SIL or CC, genetic factors appear to play little role in cervical carcinogenesis. The risk for cervical neoplasia due to HPV infection increased after marriage in Japan, suggesting a role for husbands as carriers of HPV transmission. Protection from high‐risk HPV infection may be of greatest importance for prevention of cervical cancer.

Differential expression of nitric oxide synthase isoforms in form-deprived chick eyes

PURPOSEnTo clarify whether nitric oxide synthase (NOS) is involved in development of myopia, we examined the influence of form deprivation on the expressions of NOS isoform mRNA.nnnMETHODSnNOS isoform cDNAs were amplified from total RNA extracted from control and 7-day-form-deprived chick retina-RPE (retinal pigment epithelium)-choroid, using competitive RT-PCR (reverse-transcription polymerase chain reaction). Each NOS isoform protein was also analyzed by Western blotting and immunohistochemistry.nnnRESULTnExpression of inducible NOS (iNOS) mRNA was highest in the control chick retina-RPE-choroid, followed by the expression of brain NOS (bNOS) mRNA. Expression of endothelial NOS (eNOS) mRNA was faint. The iNOS protein level, however, was only slightly higher than the levels of the bNOS and eNOS proteins and was found mainly in the outer part of the photoreceptor layer and inner and outer parts of RPE and choroid. bNOS alone was found in the outer nuclear layer. Although form deprivation reduced the iNOS and bNOS mRNA expressions, only the iNOS protein showed significant reduction.nnnCONCLUSIONnAll three NOS isoforms were expressed in chick retina-RPE-choroid. Predominant expression of iNOS, instead of bNOS and eNOS, suggested the existence of ocular tissue-specific regulation of the iNOS gene. In addition to differences in expression level, bNOS displayed regional differential expression. Moreover, only iNOS was reduced in response to form deprivation. It is suggested that NOS isoforms may be differentially involved in the mechanisms regulating the posterior eye tissues, including myopic eye growth.

The interaction of aluminium with soluble protein kinase C from mouse brain

The interaction of aluminium ion species with soluble protein kinase C, Ca2+/phospholipid-dependent protein kinase, from mouse brain has been examined in vitro. The activity of protein kinase C was increased by addition of Ca2+ displaying an EC50 value of 10.3±1.1 × 10−6 M. The A1 species inhibited the activity with an IC50 values of 8.6±0.5 × 10−5 M and 2.7±0.3 × 10−5 M in the presence of 0.5 mM Ca2+ and absence of Ca2+, respectively. Concerning the EC50 for Ca2+ activation, this was increased by the A1 species in a dose-dependent manner. Moreover, the inhibition was of a non-competitive type with respect to H1 histone and of a mixed type with respect to ATP. It is likely that the inhibition was caused by 1) the blocking of Mg2+ binding to ATP, 2) the blocking of Ca2+ binding to protein kinase C. Our results suggested that protein kinase C was involved in neurotoxicity of A1.

Behavioral abnormalities and apoptotic changes in neurons in mice brain following a single administration of allylnitrile

Abstract A single dose of allylnitrile in mice might induce persistent behavioral abnormalities, of which the mechanism is not yet known. The present study was undertaken to explore the relationship between behavioral abnormalities and pathological changes in the brain of mice following exposure to allylnitrile. Exposure to allylnitrile (63, 84, and 112u2009mg/kg, p.o.) resulted in dose-dependent changes in behavioral abnormalities, including increased locomotor activity, circling, retropulsion, head twitching, and alteration in reflexive behavior, which appeared at day 2 postdosing and were persistent throughout the experimental period (60 days) at the higher dose levels. Allylnitrile produced neuronal retraction including hyperchromasia of the nuclei in the raphe nuclei, cerebral cortex, hypothalamus, hippocampal CA1 and dentate gyrus later than 30 days. No gliosis was observed in these regions. Not all but a significant number of neurons in the hippocampal CA1, medial habenula and raphe nuclei were immunoreactive to CPP32 (Caspase-3) even at day 2. These neurons were also positive to Hoechst 33258 staining, indicating allylnitrile caused apoptotic changes in specific neurons when neuronal behaviors became apparent. These apoptotic changes were persistent even in the area without neuronal contraction such as medial habenula. However, almost all neurons in these areas were also positive to terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling (TUNEL). It is conceivable that allylnitrile caused apoptotic changes in neurons but did not always lead them to cell death immediately. Moreover, even when neuronal contraction resulted in retention of behavioral abnormalities, onset of these abnormalities seems to be associated with the impairment in the habenulo-raphe relay due to activation of apoptotic cascade in neurons.

Endogenous adenosine facilitates neurotransmission via A2A adenosine receptors in the rat superior colliculus in vivo

The concentration of endogenous adenosine in the cerebrospinal fluid increased 2-3-fold of the original level in the area of rat superior colliculus after the intraperitoneal administration of an adenosine deaminase inhibitor, EHNA (erythro-9-(2-hydroxy-3-nonyl)adenosine, 10 mg/kg). Potentials evoked in the superior colliculus by optic tract stimulation were also facilitated by 120-160% of their initial amplitudes. A selective A1 adenosine receptor antagonist, DPCPX (8-cyclopentyl-1,3-dipropylxanthine), failed to reduce such EHNA-induced facilitation. However, a selective A2A adenosine receptor antagonist, KF17837 (8(3,4-dimethoxystyryl)-1,3-dipropyl-7-methylxanthine) completely eliminated the facilitatory effects of EHNA. Northern blot analysis demonstrated abundant expression of A1 adenosine receptor mRNA in the superior colliculus. RT-PCR analysis was able to detect the concomitant expression of A2A adenosine receptor mRNA, but at levels lower than one-tenth of the striatal expression. In the superior colliculus, A2A adenosine receptors function predominantly on the facilitatory effects of adenosine, irrespective of the ubiquitous expression of A1 adenosine receptors.

Evidence for the neurotransmitter role of norepinephrine in the ventral thalamic nucleus of the guinea pig: localization, uptake and release

The localization, uptake and release of norepinephrine (NE) by the isolated guinea pig ventral thalamic nucleus (VT) were studied to determine the possible neurotransmitter role of this catecholamine. The NE content in the VT was 356 +/- 24 ng/g wet weight and intraneuronal localization of NE was demonstrated in the VT by histofluorescence. [3H]NE accumulation into the VT slices was about 3 times that of [3H]NE (10(-7) M) in the medium 10 min after incubation. Kinetic analyses indicated 2 components of [3H]NE accumulation, one representing a high (Km1 4 X 10(-7) M and Vmax1 1.5 pmol/mg/10 min) and a low (Km2 1.8 X 10(-6) M and Vmax2 5 pmol/mg/10 min) affinity uptake system. Desmethylimipramine (10(-5) M) reduced the high affinity uptake of [3H]NE by 30% of the control value. Electrical stimulation of the slices increased the efflux of [3H]NE from tissues preloaded with [3H]NE, in a current- and frequency-dependent fashion. The release of [3H]NE induced by stimulation of up to 1 mA was inhibited by tetrodotoxin (10(-6) M) or Ca-free medium containing EGTA (10(-4) M). These findings provide strong evidence for the neurotransmitter role of NE in the VT.

Recent status of the medical examiner system in Japan: Demographic variation of medicolegal deaths in hyogo prefecture and uncertainty in medicolegal investigations conducted by medical practitioners

The medical examiner system has been steadily abolished in Japan. Instead, medicolegal investigations are entrusted by the police to medical practitioners, who are not permitted to perform autopsies. The necessity for the medical examiner system was assessed through inquest records in Hyogo, one of the three prefectures which still have medical examiner systems. Standardized mortality ratios (SMRs) for accidents and suicides were negatively associated with population density, being high in rural areas with a large proportion of elderly citizens, while the SMR for natural deaths was high in urbanized areas and associated with the proportion of inquests to total resident deaths. The high proportion of inquests, however, did not always mean that inquest records were of good quality. Significant differences in the quality of medicolegal investigations seemed to exist between medical examiners and medical practitioners. That is, in order to certify the cause-of-death, medical examiners performed autopsies in about half of their cases, while only 2% of medical practitioner cases were subjected to autopsies. Medical practitioners, who certified the cause-of-death as heart failure without advising an autopsy, were regularly entrusted with inquests. It is likely that the causes-of-death for medicolegal cases may be questionable since more than 85% of all medicolegal deaths were investigated by medical practitioners, which may cause inaccuracy in at least 3-7% of mortality statistics. It is necessary to educate medical practitioners concerning the importance of mortality statistics and ICD and on the validity of autopsies, in order to obtain accurate mortality statistics from medicolegal cases.

ANALYSIS OF BOVINE SELENOPROTEIN P-LIKE PROTEIN GENE AND AVAILABILITY OF METAL RESPONSIVE ELEMENT (MRE) LOCATED IN ITS PROMOTER

Selenoprotein P-like protein, similar to selenoprotein P, uses multiple TGAs for incorporation of selenocysteines but not as stop codons. It is also characterized by having a His-Pro-rich domain and a regionally differential expression pattern. Hence, in addition to selenium metabolism, this protein is considered to have a developmental function. In the present study, the structure of the selenoprotein P-like protein gene was analyzed. The gene consisted of five exons, and the 5-flanking region contained a TATA box, TCF-1-CS, bHLH-CS, gamma-IRE-CS, c-Myb-CS, C/EBP-CS, HNF-5-CS, MRE2-CS, etc. The presence of motifs like TCF-1-CS, c-Myb-CS, etc. supports the suggestion that this protein is involved in cellular maturation. Since the presence of MRE2-CS suggests that this protein is related to the antidote effect of selenium against heavy metal intoxication, the availability of this motif was examined using bovine kidney cell lines, CKT-1 and MDBK. Metallothionein mRNA markedly increased 6 h after administration of 10(-6) M CdCl2 and ZnCl2 in both cell lines. No significant alteration was observed in selenoprotein P-like protein mRNA, whereas its basal expression was high, indicating that this protein is constitutively expressed. Thus, it is still possible that this protein acts as an antidote, even though it is not inducible by heavy metals.