Effects of perinatal lipopolysaccharide (LPS) exposure on the developing rat brain; modeling the effect of maternal infection on the developing human CNS.

In the present study, we examined the effect of perinatal Escherichia coli lipopolysaccharide (LPS) exposure on the developing rat cerebellum and tested the hypothesis that maternal infections impact brain structure and function by mechanisms involving increase in oxidative stress and changes in brain type 2 iodothyronine deiodinase (D2)- and thyroid hormone (TH)-responsive genes. Spontaneously hypertensive rat (SHR) and Sprague-Dawley (SD) rat dams were challenged with LPS (200 µg/kg body weight) exposure during pregnancy (G10-G15) and lactation (P5-P10), the time periods corresponding, respectively, to the first/second and the third trimesters of human pregnancy. LPS exposure resulted in a significantly decreased motor learning in SD male (29.8 %) and in female (55.0 %) pups (p < 0.05); changes in rollover and startle response showed only a trend. The LPS challenge also resulted in a trend (p = 0.09) toward increased cerebellar levels of the oxidative stress marker 3-nitrotyrosine (3-NT) in SD male (16.2 %) and female (21.2 %) neonates, while 3-NT levels were significantly decreased (p < 0.05) in SHR female pups. D2 activity, responsible for local intra-brain conversion of thyroxine (T4) to the active hormone, 3',3,5-triiodothyronine (T3), was significantly (p < 0.05) decreased in LPS-challenged SHR male (40.3 %) and SD female (47.4 %) pups. Several genes were affected by LPS. Notably, D2 (DIO2) and brain-derived neurotrophic factor (BDNF) were significantly elevated in SHR females, while transthyretin (TTR) expression was decreased in both SD males and females (P < 0.05). In vitro chronic exposure of cerebellar cultures to LPS resulted in decreased arborization of Purkinje cells while D2 was only increased transiently. Our data demonstrate that perinatal LPS exposure impacts the developing cerebellum in strain- and sex-dependent manner via complex mechanisms that involve changes in oxidative stress, enzymes involved in maintaining local TH homeostasis, and downstream gene expression.

Risk factor analysis for low blood pressure and hyponatremia in acutely and subacutely spinal cord injured patients.

STUDY DESIGN: Case control. OBJECTIVE: To clarify the predictors of low blood pressure (BP) and hyponatremia after spinal cord injury (SCI) and to discuss their pathophysiology. SETTING: A SCI center in Japan. METHODS: Age, gender, initial ASIA impairment scale (AIS) score, BP, blood electrolytes (sodium, K and Cl) and biochemical markers were evaluated at 1 month after injury. Risk factors of low BP and hyponatremia were analyzed using uni- and multivariate logistic regression models. RESULTS: This study comprised of 172 SCI patients. Initial AIS score (Odds ratio (OR): 1.24, 95% confidence intervals (CI) 1.13-1.49, P-value <0.01) and hyponatremia (OR: 3.71, 95%CI 1.27-6.96, P<0.01) were the most important risk factors of low BP. As a second step, risk factors of hyponatremia were initial AIS score (OR: 1.36, 95%CI 1.08-2.78, P<0.01) and age (OR: 1.55, 95%CI 1.17-2.93, P<0.01). CONCLUSIONS: In acute and subacute period, the more severe SCI and lower AIS score patients have the more frequently low BP and/or hyponatremia do appear.

FcepsilonRIalpha gene (FCER1A) promoter polymorphisms and total serum IgE levels in Japanese atopic dermatitis patients.

Two promoter polymorphisms of the high-affinity IgE receptor alpha-subunit (FcepsilonRIalpha) gene (FCER1A), -66T>C (rs2251746) and -315C>T (rs2427827), were analysed in Japanese atopic dermatitis subjects. Patients with the -315CT/TT genotype tended to have higher total serum IgE levels, while the proportion of -315CT/TT genotype or the -315T allele was significantly higher in those with highly elevated total serum IgE concentrations.

Functional analysis of a polymorphism in the promoter region of the IL-12/23p40 gene.

BACKGROUND: Human IL-12B gene on chromosome 5q31 encodes the common p40 subunit of IL-12 and IL-23. IL-12 is known to play critical roles in the generation of T-helper type 1 (TH(1)) cells, whereas IL-23 is involved in maintenance and/or population expansion of TH(17) cells. Although several reports suggested an association between a polymorphism (-6415CTCTAA/GC) in IL-12B and asthma, the molecular mechanism how this polymorphism is involved in allergic inflammation is still unclear. METHODS: The transcription activity was analysed by reporter assay. A transcription factor binding to -6415 polymorphic site was identified by electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assay. The amount of cytokines produced from peripheral monocytes were determined by ELISA. RESULTS: Reporter assay showed that the transcription activity of the GC allele was higher than that of the CTCTAA allele. A transcription factor Sp1 bound to the region including the GC allele with a higher affinity than that of the CTCTAA allele in EMSA. In vivo binding of Sp1 to IL-12B gene carrying -6415GC was confirmed by ChIP assay. Overexpression of Sp1 up-regulated transcription activity of promoter carrying GC allele sequence, whereas the CTCTAA promoter was not affected by Sp1. We examined the correlation between -6415CTCTA/GC polymorphism and production of cytokine IL-12/23p40, IL-12p70, and IL-23 on peripheral blood monocytes, and monocytes with the GC/GC allele exhibited significantly higher expression of IL-12p70 protein than those with the CTCTAA/CTCTAA allele (P=0.009). CONCLUSIONS: The -6415 polymorphism is involved in cytokine production potential by affecting Sp1-mediated transcription activity.

Morphological transition and emulsification failure in globular microemulsions.

We consider the condensation transition of microemulsion droplets of oil which are dispersed in water in the presence of surfactant. Since a macroscopic oil phase is formed due to this transition, it is called "emulsification failure." Based on the free energy approach, we determine the transition lines between the spherical and the cylindrical droplet phases as well as the phase boundary lines of the emulsification failure. The phase diagrams are calculated by changing the physical properties of the surfactant monolayer such as the saddle-splay modulus and the spontaneous curvature. For a negative saddle-splay modulus, the spherical droplet phase coexists with the excess oil phase. In some cases, a re-entrant transition (sphere-->cylinder-->sphere) is expected to take place. For a positive saddle-splay modulus, the system undergoes a direct transition from the cylindrical droplet phase to the macroscopically phase separated state. The sphere-to-cylinder transition line approaches the emulsification failure boundary as the saddle-splay modulus becomes larger.

Size-dependent uptake of electrically neutral amphipathic polymeric nanoparticles by cell-sized liposomes and an insight into their internalization mechanism in living cells.

Size-dependent uptake behaviors of electrically neutral amphipathic polymeric nanoparticles in cell-sized liposomes and living cells were investigated. Kinetic analyses and the particle size distribution suggested a size-dependent penetration mechanism (size threshold: 3.1 nm). The definite size-dependent uptake provides a new insight into the interactions between nanomaterials and living cells.

Porcine growth hormone: molecular cloning of cDNA and expression in bacterial and mammalian cells.

Porcine growth hormone (PGH) precursor cDNAs were cloned from a pituitary cDNA library constructed in lambda gt11 by immunoscreening. One of the three clones characterized contained an entire nucleotide sequence for the 216-amino-acid precursor molecule. The deduced amino-acid sequence of PGH confirmed the sequence previously reported for that of the genomic DNA of PGH except for one base difference in the coding sequence. Expression of the full-length PGH cDNA was achieved in bacteria and mammalian cells. The mammalian cell line, COS-1, produced the GH molecule which processed the signal peptide and had the same molecular weight as standard PGH, in contrast to the higher molecular weight of the bacterial product. Radioimmunoassay of the recombinant PGH produced in COS-1 cells also revealed an inhibition curve similar to that of the standard PGH.

MafG-2 is a novel Maf protein that is expressed by stimulation of extracellular H(+).

We cloned MafG-2, a novel splice variant of MafG, from rat brain by RT-PCR method. MafG-2 differs from the previously published MafG by an insertion of 27 amino acids. Sequence analysis of the cDNA-encoded MafG-2 showed that MafG-2 contains basic domain and basic leucine zipper (bZip) motif. Transient transfection studies with GFP-MafG-2 chimera protein indicate that MafG-2 is localized in the nuclei of transfected COS-7 cells. To determine whether gene expression of mafG-2 mRNA is induced by an increase in extracellular protons, we analyzed expression of the mRNA in PC12 cells after an increase in extracellular proton concentration. We found that the mafG-2 mRNA expression increased when extracellular pH was decreased gradually from 7.40 to 7.20 and that there was a significant correlation between extracellular pH value and the expression of mafG-2 mRNA. These results suggest that an increase in extracellular proton may induce the expression of mafG-2 mRNA and MafG-2 may be involved in signal transduction of extracellular of H(+).

Extracellular H+ stimulates the expression of c-fos/c-jun mRNA through Ca2+/calmodulin in PC12 cells.

Evidence has accumulated that an increase in extracellular protons stimulates the transmembrane mechanism to induce various intracellular responses, such as the expression of c-fos and c-jun. In the present study, we aimed to obtain evidence that an increase in extracellular protons induces expression of c-fos/c-jun mRNA in PC12 pheochromocytoma cells of rats. We found that the c-fos/c-jun mRNA expression increased when extracellular pH was decreased gradually from 7.40 to 7.20 and that there was a significant correlation between extracellular pH values and the expression of c-fos/c-jun mRNA. To determine whether the Ca2+/calmodulin system subserves the H+-induced expression of c-fos/c-jun, Ca2+/calmodulin inhibitor trifluoperazine was added to PC12 cells. We found that trifluoperazine inhibited the expression of the H+-induced c-fos/c-jun mRNA by 30-35%. In contrast, trifluoperazine did not inhibit the expression of phorbol-induced c-fos/c-jun mRNA. These results indicate that an increase in extracellular protons induces the expression of c-fos/c-jun mRNA, and this expression is mediated partly by the Ca2+/calmodulin system.

Molecular cloning and sequencing of the cDNA coding for a calcium-binding protein regucalcin from rat liver.

The cDNA of a Ca(2+)-binding protein regucalcin was cloned from a rat liver cDNA library which was constructed in lambda ZAPII by immunoscreening. Positive clones were obtained from which spanned the region of interest, and they gave a sequence of 1.7 kb by sequencing with the dideoxynucleotide method. Analysis of the sequence of the cloned cDNA showed that the cDNA encoded the complete amino acid sequence of the regucalcin molecule. Regucalcin was composed of 299 amino acid residues and its molecular weight was estimated to be 33,388 Da. The hydropathy profile of regucalcin showed a highly hydrophilic character. The nucleotide and amino acid sequences of regucalcin did not have statistically significant homology, as compared with the registered sequences which are found in the EMBL and GenBank databases containing several other Ca(2+)-binding proteins (calmodulin, calbindin-D28k and S-100 beta). The regucalcin molecule did not contain the EF-hand motif as a Ca(2+)-binding domain. The present study demonstrates that regucalcin is a unique Ca(2+)-binding protein in the liver of rats.

Expression of hepatic calcium-binding protein regucalcin mRNA is mediated through Ca2+/calmodulin in rat liver.

The effect of signal transduction-related factors was investigated to clarify the expression mechanism for mRNA of the hepatic Ca(2+)-binding protein regucalcin in the liver of rats. The change of regucalcin mRNA levels was analyzed by Northern blotting using liver regucalcin cDNA (0.6 kb). A single intraperitoneal administration of calcium chloride (15 mg Ca2+: 0.374 mmol/100 g body weight) to rats induced a remarkable increase of regucalcin mRNA in liver; the level was about 170% of controls at 30 min after administration. This increase was completely inhibited by simultaneous administration of trifluoperazine (5.0 mg/100 g), an antagonist of calmodulin. On the other hand, a single intraperitoneal administration of phorbol ester or dibutyryl cAMP (10-1,000 micrograms/100 g) did not cause a significant alteration of hepatic regucalcin mRNA levels. Also, administration of zinc, copper and cadmium (0.374 mmol of metal ion/100 g) did not have an appreciable effect on hepatic regucalcin mRNA levels. These findings demonstrate that the expression of hepatic regucalcin mRNA is mediated through Ca2+/calmodulin.

Calcium administration stimulates the expression of calcium-binding protein regucalcin mRNA in rat liver.

The distribution and expression of mRNA encoding the Ca(2+)-binding protein regucalcin in rats were investigated by Northern blot analyses. Liver regucalcin cDNA (0.6 kb) was used as a probe. The analyses of total RNAs extracted from various tissues of rat indicated that regucalcin mRNA was mainly present in liver but only slightly in kidney with a size of 1.8 kb. The expression level decreased with increasing age (3, 10 and 25 weeks). A single intraperitoneal administration of calcium chloride (15 mg Ca/100 g body weight) induced a remarkable increase in regucalcin mRNA in liver; the level was about 200% of control at 30 min after the administration. Subsequently, the expression level began to decrease with time and was about 40% of control level at 120 min after the administration. The increase in regucalcin mRNA levels at 30 min after calcium administration was dose-dependent. These observations show that the expression of regucalcin mRNA is specific in liver of various tissues, and that it is regulated by Ca2+ administration. Regucalcin may have a role as regulatory protein for calcium homeostasis in liver cells.

Detection of living cells that express AP1 using a fluorolabeled DNA probe.

Activator protein 1 (AP1) is a complex of Fos and Jun, and it regulates the transcription of genes possessing the AP1-binding sequence. The purpose of this study was to detect living cells that express AP1 after stimulation with a tumor promoter. The Fos and Jun components of AP1 were induced rapidly and transiently in PC12 cells following the addition of phorbol ester (phorbol 12-myristate 13-acetate, PMA). The DNA fragment containing the AP1-binding sequence was combined with ethidium bromide, which was used as a fluorescent probe. The probe was transfected into the cells using cationic liposomes. Fluorescence in the transfected cells was observed using a fluorescence microscope. The nuclei of transfected cells emitted strong fluorescence in the presence of PMA, whereas weak fluorescence was retained in the cytoplasm in its absence. The former phenomenon is evidence that AP1 combined with the fluorescent probe was transported into the nuclei. This study suggests that such a fluorolabeling method is feasible to detect living AP1-expressed neurons.

Studies on analgesic agents. 1.1a Preparation of 1,2-diphenyl-2-(4-substituted 1-piperazinyl)ethanol derivatives and structure-activity relationships.

The preparation and analgesic activity of a series of the title compounds (8-55 and 57) are described. The intermediates, 2-phenyl-2-(1-piperazinyl)acetophenones 5 and 6, were prepared from benzyl phenyl ketones 3 via their bromides 4. On reduction, compounds 5 afforded the titled compounds 8-12, 16, and 26-48. Compounds 13-15 and 17-25 were obtained by alkylation or benzylation of 1.2-diphenyl-2-(1-piperazinyl)ethanols 7 derived from 6 by reduction. The reduction of 5 and 6 with metal hydrides predominantly gave the erythro isomers. The erythro isomers were remarkably more active than their threo isomers. The more active members in this series of compounds were 16 and derivatives 35 and 37-44 of dl-erythro-1-phenyl-2-(substituted phenyl)-2-[4-(p-methoxybenzyl)-1-piperazinyl]ethanol. Compounds 16, 43, and 44 were the most active with a potency of about two to three times that of codeine. Racemates 16 and 38 were resolved into their optical isomers and it was found that (-)-16 and (+)-38 were more potent than their antipodes. Structure-activity relationship are discussed.

In vitro study of H+-sensitive neurons in the ventral medullary surface of neonate rats.

We hypothesized that the direct stimulus of the central chemoreceptor neurons is the CO2/H+-induced change in intracellular pH (pHi). If it is true, pHi responses during hypercapnic stimulation should be exhibited in the central chemoreceptor neurons in the ventral medullary surface (VMS) and some neurons in the CO2/H+ sensitive regions such as the nucleus tractus solitarii of the medial dorsal medulla (MDM). To test this hypothesis, the cultured VMS and MDM neurons (control) derived from one day-old neonate rats were labeled with H+-sensitive fluorescent indicator 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF), and were exposed to perfusate of various pHs. The H+-sensitive neurons were determined by a rapid decrease in the intracellular BCECF fluorescence intensity. In almost all the MDM neurons (99.6%) and 94% of the VMS neurons, the intracellular BCECF fluorescence intensity remained unchanged when the extracellular pH (pHo) was decreased. In contrast, in 0.4% of the MDM neurons (8/1800) and in 6% of the VMS neurons (111/1800), the intracellular BCECF fluorescence intensity decreased when the pHo was decreased from 7.4 to 7.2. This subpopulation of MDM and VMS neurons were considered to be H+-sensitive neurons. The H+-sensitive neurons in the VMS showed positive immunoreactivity to glutamate (57%, 17/30) and glutamic acid decarboxylase (23%, 7/30), but no immunoreactivity to choline acetyltransferase, tyrosine hydroxylase, phenylethanolamine N-methyltransferase, somatostatin, serotonin and substance P. These results indicate that the H+-sensitive neurons are present specifically in the VMS, and are mainly glutamatergic and GABAergic.

Molecular cloning of Rhombex-40 a transmembrane protein from the ventral medullary surface of the rat brain by differential display.

Respiration-related neurons, which detect various chemicals in cerebrospinal fluid, are localized to the ventral medullary surface (VMS). We hypothesized that expression of genes involved in respiratory function is upregulated in the VMS. By differential display, we looked for genes differentially expressed in VMS neurons and cerebral cortical neurons. Seventeen clones of interest were isolated, and sequence analysis revealed that one of these clones encoded a putative transmembrane protein, rhombencephalic expression protein-40 kDa (Rhombex-40). The rat Rhombex-40 was composed of 374 amino acid residues, and the predicted secondary structure displays a signal peptide in the N-terminus and single-pass transmembrane domain in the center of the sequence. An analysis of consensus sequences identified several phosphorylation sites in the intracellular domain. Expression of rat Rhombex-40 mRNA is high in the brain, and low in lung, liver and kidney. No homologous protein sequence was found in database searches. Whereas the biological function of this protein is presently unknown, its structural features and high expression in the brain suggest that Rhombex-40 may function as a novel transmembrane molecule in neural cells of the brain.

Spontaneous spinal subarachnoid hematoma--case report.

BACKGROUND: Spinal subarachnoid hemorrhage is unusual, and rarely results in spinal subarachnoid hematoma because the cerebrospinal fluid tends to dilute the blood and prevent the formation of clots. We describe a patient with spinal subarachnoid hematoma of unusual spontaneous origin. CASE: A 66-year-old female presented with sudden onset of intense back pain with paraplegia. Magnetic resonance imaging demonstrated a mass lesion between T2 and T6, compressing the spinal cord anteriorly. Emergency osteoplastic laminotomy exposed a hematoma in the subarachnoid space from T2 to T6, but no source of the hemorrhage was found. The patient was able to walk by herself about 20 days after the operation. CONCLUSION: The outcome is significantly influenced by the duration between onset and operation, preoperative neurologic status, and rapidity of symptom progression. Therefore, we emphasize the importance of early diagnosis, and rapid and complete operative removal of spinal subarachnoid hematoma in order to achieve the best outcome.

Altered cerebellum development and dopamine distribution in a rat genetic model with congenital hypothyroidism.

Thyroid hormones play crucial roles in the development and functional maintenance of the central nervous system. Despite extensive studies of the neural function of thyroid hormones, little is known about the effects of hypothyroidism on behavioural traits and the mechanisms underlying such effects. In the present study, we report an investigation of congenitally hypothyroid mutant rdw rats, revealing a novel function of thyroid hormones in the central nervous system. The rdw rats were subjected to behavioural analyses such as the rotarod test, open field test and circadian activity measurement. To determine the cause of behavioural disorders, cerebellar morphogenesis was examined by immunohistochemical analysis, and the axonal transport of dopamine in the nigrostriatal pathway was analysed by high-performance liquid chromatography and western blotting. The effects of thyroxine administration to the rdw rats were examined by behavioural analysis. The rdw rats showed severe impairment of motor coordination and balance. This could be explained by the fact that the rats showed severe retardation of cerebellar morphogenesis, which correlates with the small somata and poor dendritic arborisation of Purkinje cells and retarded migration of granule cells particularly during the first two postnatal weeks. Moreover, the rdw rats showed hypoactivity, characterised by decreased circadian locomotor activity. After weaning, thyroxine administration improved the dwarfism in rdw rats but had no effect on cerebellar function. In addition, the rdw rats showed anxiety and depression intrinsically to novel surroundings. Interestingly, the rdw rats showed high levels of dopamine in the substantia nigra and low levels in the striatum, an important centre for the coordination of behaviour. Furthermore, low levels of tubulin in the striatum were detected, indicating the aberrant axonal transport of dopamine in the nigrostriatal pathway as a result of the reduced delivery of microtubules. These findings indicate an important function of thyroid hormones in cerebellar formation and in the regulation of axonal transport of dopamine. Moreover, rdw rats will be useful for studies of brain function and behavioural disorders in congenital hypothyroidism.