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.

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.

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.

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.

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.

The phase behavior of mixed lipid membranes in the presence of the rippled phase.

We propose a model describing liquid-solid phase coexistence in mixed lipid membranes by including explicitly the occurrence of a rippled phase. For a single component membrane, we employ a previous model in which the membrane thickness is used as an order parameter. As function of temperature, this model properly accounts for the phase behavior of the three possible membrane phases: solid, liquid and the rippled phase. Our primary aim is to explore extensions of this model to binary lipid mixtures by considering the composition dependence of important model parameters. The obtained phase diagrams show various liquid, solid and rippled phase coexistence regions, and are in quantitative agreement with the experimental ones for some specific lipid mixtures.

Tension-induced morphological transition in mixed lipid bilayers.

Recently, Rozovsky et al. reported on the morphology and dynamics of superstructures in three-component lipid bilayers containing saturated lipid, unsaturated lipid, and cholesterol (Rozovsky, S.; Kaizuka, Y.; Groves, J. T. J. Am. Chem. Soc. 2005, 127, 36). We suggest that the observed sequence of the striped-to-hexagonal morphological transition in mixed bilayers can be attributed to an enhanced membrane surface tension that is induced by the vesicle adhesion onto the solid surface.

Unbinding and preunbinding in surfactant solutions.

We propose models for the first-order unbinding transition of lyotropic lamellae in surfactant solutions. The coupling between the surfactant volume fraction and the elastic degree of freedom is considered so that the net attractive interaction between the surfactant molecules is enhanced. The elastic degree of freedom can be either (i) a membrane elastic degree of freedom or (ii) a bulk elastic degree of freedom. The phase behaviors of these two models are analyzed. For both cases, the unbinding transition becomes first order when the coupling is strong enough. We determine the associated preunbinding line which separates two lamellar phases having different repeat distances.

Importance of transmembrane segments in Escherichia coli SecY.

To assess the functional importance of the transmembrane regions of SecY, we constructed a series of SecY variants, in which the six central residues of each transmembrane segment were replaced by amino acid residues from either transmembrane segment 3 or 4 of LacY. The SecY function, as assessed by the ability to complement cold-sensitive secYmutants with respect to their growth and translocase defects, was eliminated by the alterations in transmembrane segments 2, 3, 4, 7, 9 and 10. Among them, those in segments 3 and 4 had especially severe effects. In contrast, transmembrane segments 1, 5, 6, and 8 were more tolerant to the sequence alterations. The purified protein with an altered transmembrane segment 6 retained, in large measure, the ability to support SecA-dependent preprotein translocation in vitro. These results will help us to further understand how the SecYEG protein translocation channel functions.

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(+).

Rhombex-29, a novel gene of the PLP/DM20-M6 family cloned from rat medulla oblongata by differential display.

The ventral medullary surface (VMS) is known as the site of the central chemosensitive neurons. These neurons sense excess CO(2)/H(+) dissolved in the cerebrospinal fluid that superfuses the VMS and induce hyperventilation. We hypothesized that genes specific for hyperventilation are expressed much more highly in VMS neurons than in extra-VMS neurons in other parts of the central nervous system (CNS). Applying the differential display technique to the brain of adult rats, we differentiated the mRNAs of the VMS neurons from those of cerebral cortex neurons. Seventeen candidate clones were selected, and their sequences were analyzed. Among these 17 clones, one encodes a novel four-transmembrane protein, which we named rat Rhombex-29. Structural analysis and the phylogenic tree showed that rat Rhombex-29 is homologous to the major CNS myelin protein PLP/DM20-M6 family and belongs to the intermediate type between mouse M6b and shark DMgamma. As the embryos grew into adult rats, constant expression of rat Rhombex-29 mRNA was found in the brain. Hypercapnic stimulation increased expression of rat Rhombex-29 mRNA in the VMS neurons but not in the cerebral cortex neurons. These results indicate that the VMS neurons are endowed with a novel gene, rat Rhombex-29, that is sensitive to H(+).

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.

Cloning of MafG homologue from the rat brain by differential display and its expression after hypercapnic stimulation.

The ventral medullary surface (VMS) is a site of the medullary chemoreceptor neurons which sense excess protons (H+) derived from hypercapnia and facilitate respiration. We hypothesized that expression of genes involved in H+-sensitivity is higher in the VMS than in other central nervous system areas. By using the differential display technique, we differentiated the mRNAs of VMS neurons from those of cerebral cortical neurons. Seventeen clones of interest were isolated, and sequence analysis revealed that one of these clones had an encoding nuclear transcription factor, MafG. MafG is a member of Maf protein family, and the founding member of the family (v-Maf) was originally discovered as the transduced transforming component of avian musculoaponeurotic fibrosarcoma virus, AS42. The rat MafG was composed of 162 amino acid residues and was conserved among the primary structures of various species. Expression of rat mafG mRNA is high in the VMS, heart and skeletal muscle while the cerebral cortex, cerebellum, liver, stomach and intestine show moderate expression. To determine whether the expression of mafG mRNA is induced by hypercapnic stimulation, 7% CO2 in air was inhaled to rats for 5 min. We found that the hypercapnic stimulation induced the gene expression of mafG. These results suggest that MafG may be involved in H+-sensitivity and respiratory regulation in the VMS.

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.

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.

The 5' end sequences and exon organization in rat regucalcin gene.

The 5'-flanking region of the gene for a Ca(2+)-binding protein regucalcin was cloned from a rat genomic library which was constructed in lambda EMBL3 SP6/T7 vector. The genomic library was screened by using the radiolabeled probe with the 5' region (0.5 kb) of rat regucalcin complementary deoxyribonucleic acid (cDNA). Positive clone had the 5.5 kb fragment which was hybridized with the 5'-probe. This fragment contained three exons (I-III) of the gene coding for a rat regucalcin. The nucleotide sequence of exons completely agreed with that of a rat regucalcin cDNA clone. A supposed translational initiation site existed in the exon II. Homology analysis showed that a putative transcription start site in the rat regucalcin gene was located at position 26 downstream from a TATA-box. Another upstream element, a CCAAT box-like sequence, was located at -170. Moreover, there were many regulatory elements (Hox, AP-1, AP-2 and AP-4) in the 5'-flanking region of the rat regucalcin gene. The organization of rat regucalcin gene seemed to be about 18 kb in size and consisted of seven exons and six introns.

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.

Genomic cloning and chromosomal assignment of rat regucalcin gene.

The gene for a Ca(2+)-binding protein regucalcin was cloned from a rat genomic library which was constructed in lambda FIX II by screening with radiolabeled probe (complementary DNA of rat liver regucalcin). Positive clone had 19.9 kb insert of size and contained four exons of the gene coding for a rat regucalcin. These exons included the partial coding sequence (61.2% of open reading frame) and the entire 3'-untranslated region of the gene. The nucleotide sequence of exons completely agreed with that of a rat regucalcin cDNA clone. The sequence analysis of the clone showed that the identifier sequence and two simple repeated sequences exist in the intron of the gene. Moreover, chromosomal location of the rat regucalcin gene was determined by direct R-banding fluorescence in situ hybridization (FISH) method with the 19.9 kb clone containing four exons. The regucalcin gene was localized on rat chromosome Xq11.1-12 proximal end.