Transient expression of neuropeptide W in postnatal mouse hypothalamus--a putative regulator of energy homeostasis.

Neuropeptide B and W (NPB and NPW) are cognate peptide ligands for NPBWR1 (GPR7), a G protein-coupled receptor. In rodents, they have been implicated in the regulation of energy homeostasis, neuroendocrine/autonomic responses, and social interactions. Although localization of these peptides and their receptors in adult rodent brain has been well documented, their expression in mouse brain during development is unknown. Here we demonstrate the transient expression of NPW mRNA in the dorsomedial hypothalamus (DMH) of postnatal mouse brain and its co-localization with neuropeptide Y (NPY) mRNA. Neurons expressing both NPW and NPY mRNAs begin to emerge in the DMH at about postnatal day 0 (P-0) through P-3. Their expression is highest around P-14, declines after P-21, and by P-28 only a faint expression of NPW and NPY mRNA remains. In P-18 brains, we detected NPW neurons in the region spanning the subincertal nucleus (SubI), the lateral hypothalamic (LH) perifornical (PF) areas, and the DMH, where the highest expression of NPW mRNA was observed. The majority of these postnatal hypothalamic NPW neurons co-express NPY mRNA. A cross of NPW-iCre knock-in mice with a Cre-dependent tdTomato reporter line revealed that more than half of the reporter-positive neurons in the adult DMH, which mature from the transiently NPW-expressing neurons, are sensitive to peripherally administrated leptin. These data suggest that the DMH neurons that transiently co-express NPW and NPY in the peri-weaning period might play a role in regulating energy homeostasis during postnatal development.

GABAA receptor-mediated input change on orexin neurons following sleep deprivation in mice.

Orexins are bioactive peptides, which have been shown to play a pivotal role in vigilance state transitions: the loss of orexin-producing neurons (orexin neurons) leads to narcolepsy with cataplexy in the human. However, the effect of the need for sleep (i.e., sleep pressure) on orexin neurons remains largely unknown. Here, we found that immunostaining intensities of the α1 subunit of the GABAA receptor and neuroligin 2, which is involved in inhibitory synapse specialization, on orexin neurons of mouse brain were significantly increased by 6-h sleep deprivation. In contrast, we noted that immunostaining intensities of the α2, γ2, and ß2/3 subunits of the GABAA receptor and Huntingtin-associated protein 1, which is involved in GABAAR trafficking, were not changed by 6-h sleep deprivation. Using a slice patch recording, orexin neurons demonstrated increased sensitivity to a GABAA receptor agonist together with synaptic plasticity changes after sleep deprivation when compared with an ad lib sleep condition. In summary, the GABAergic input property of orexin neurons responds rapidly to sleep deprivation. This molecular response of orexin neurons may thus play a role in the changes that accompany the need for sleep following prolonged wakefulness, in particular the decreased probability of a transition to wakefulness once recovery sleep has begun.

Comparison of time-action profiles of insulin glargine and NPH insulin in normal and diabetic dogs.

Intermediate insulin injections are commonly used for glycemic control in insulin dependent diabetic dogs acting as a replacement for natural insulin. Neutral Protamin Hagedorn (NPH) insulin and insulin glargine are two types of injectable insulin preparations commonly used in humans. In our study, we investigated the time-action profiles of both aforementioned insulin preparations in normal dogs in order to determine whether co-administration of NPH and glargine would be of benefit to insulin dependent diabetic dogs as it is for humans suffering from insulin dependent diabetes. Time-action profiles of NPH insulin and insulin glargine in normal dogs demonstrated a clear difference between both insulin preparations confirming that NPH insulin is an intermediate-acting preparation whereas insulin glargine is a long-lasting preparation. In addition, co-administration of NPH insulin and insulin glargine resulted in tight glycemic control as compared to NPH insulin alone in insulin dependent diabetic dogs. However, co-administration result in hypoglycemia at the dosages tested.

Universal GFP reporter for the study of vascular development.

We report the generation and characterization of transgenic mouse and zebrafish expressing green fluorescent protein (GFP) specifically in vascular endothelial cells in a relatively uniform fashion. These reporter lines exhibit fluorescent vessels in developing embryos and throughout adulthood, allowing visualization of the general vascular patterns with single cell resolution. Furthermore, we show the ability to purify endothelial cells from whole embryos and adult organs by a single step fluorescence activated cell sorting. We expect that these transgenic reporters will be useful tools for imaging vascular morphogenesis, global gene expression profile analysis of endothelial cells, and high throughput screening for vascular mutations.

Identification of a potent neurotrophic substance for ciliary ganglionic neurons in fetal calf serum as insulin-like growth factor II.

When fetal calf serum (FCS) alone is used as a trophic support for cultured chicken parasympathetic ciliary ganglionic (cCG) neurons, it does not show any survival-promoting effects on these neurons. When FCS is applied to heparin-affinity chromatography, however, potent survival-promoting activity is obtained in the fraction eluted with 0.5 M NaCl. Using cCG neurons as a bioassay system, this neurotrophic activity was purified by a combination of heparin-affinity chromatography, gel filtration chromatography, and Sep-Pak C18 cartridge. The 40-50-kDa fractions from the gel filtration column with strong survival-promoting activity were shown to contain insulin-like growth factor II (IGF-II) by immunoblot analysis. By acidification, the survival-promoting activity and IGF-II were translocated together from the 40-50-kDa to the 7-10-kDa fractions, and the survival-promoting activity in the 7-10-kDa fractions was blocked by an anti-IGF-II neutralizing monoclonal antibody. These results indicate that the neurotrophic substance in 0.5 M NaCl-eluate from heparin-affinity chromatography is IGF-II and that mechanisms may exist in vivo for the activation of latent IGF-II, whose biological effects may be blocked by its specific binding proteins.

An observation on subsurface defects of ultra high molecular weight polyethylene due to rolling contact.

Ultra high molecular weight polyethylene (UHMWPE) has been used in artificial joints for a few decades, and wear of UHMWPE has been one of the main problems. Though many other materials have been tested over the years, the best clinical results are still achieved with UHMWPE. This makes the study of UHMWPE, especially in relation to artificial joints, very important. Frequently, more severe wear can be observed in artificial knee joints than in artificial hip joints especially when the flaking-like wear occurs. This flaking-like wear can lead to significant destruction of the artificial knee joint. Macroscopically, artificial knee joints have combinational movements of rolling and sliding in order to simulate the motion of the normal knee joint. The components of motion are separated to make study easier. Fatigue tests of UHMWPE under the rolling contact condition were performed in this study. Three ceramic spheres were rolled over the UHMWPE specimen using 37 degrees C distilled water as a lubricant. The UHMWPE specimen was observed by the scanning acoustic tomography, microscopy, and SEM. Some subsurface defects could be observed by SAT even before experiments. Although the apparent wear is not observed on the surface, there was an increase in the number of observable subsurface cracks in the UHMWPE specimen. This shows that cracks occur under the surface after a 10(7) rolling contact loading, which is very close to the cyclic loading and unloading with very little friction compared to the sliding contact.

Induction of phosphatidic acid by fibroblast growth factor in cultured baby hamster kidney fibroblasts.

Basic fibroblast growth factor (bFGF/FGF-2) is a strong mitogenic inducer of cultured baby hamster kidney (BHK) cells. When cultured BHK cells were stimulated with FGF-2, phosphatidic acid (PA) was induced within 2 min, peaked at 5 min and gradually decreased. Phospholipase D (PLD) was also mitogenic for cultured BHK cells and this effect was mediated via PA. The possibility that PA induction by FGF-2 is an essential signaling step for BHK cell proliferation is discussed.

Expression of smg p25A/rab 3A guanine nucleotide dissociation inhibitor (GDI) in neurons and glial cells from rat brain.

smg p25A/rab 3A is a member of the rab family of small guanosine 5'-trisphosphate (GTP)-binding proteins, which are postulated to act as specific regulators of membrane trafficking in exocytosis and endocytosis. In neural tissues, smg p25A/rab 3A is expressed only in neurons but not in glia. In this study, expression of a regulatory protein for smg p25A/rab 3A, guanine nucleotide dissociation inhibitor (smg p25A/rab 3A GDI), in neurons and glia isolated from rat brain was examined. The smg p25A/rab 3A GDI mRNa was detected as early as on gestational day 14 in the fetal brain, and its level reached a maximum 5 days after birth. By immunoblot analysis, smg p25A/rab 3A GDI was detected in the homogenate of rat hippocampus as well as in that of astrocytes in primary culture. By immunocytochemical analysis, the smg p25A/rab 3A GDI immunoreactivity was detected in hippocampal neurons and oligodendrocytes. The immunoreactivity of astrocytes was much lower than that of neurons and oligodendrocytes. These results indicate that smg p25A/rab 3A GDI may play a fundamental role in membrane trafficking both neurons and glia by interacting with multiple rab family proteins.

Expression and localization of smg p25A (= rab3A) in cultured rat hippocampal cells.

We have studied the expression of smg p25A and synaptophysin in cultured hippocampal neurons isolated from 5-day-old rat brain by an immunocytochemical technique. In a dispersed cell culture seeded on astrocyte monolayers, well-branching neurite proliferation was observed along with age in culture. The synaptophysin immunoreactivity was present in the neuronal cell bodies and neurites at 1 and 5 days in vitro (DIV) and was eventually localized to discrete areas along neurites at 15 DIV while the immunoreactivity in cell bodies became less prominent. On the other hand, the smg p25A immunoreactivity was observed in the neuronal cell bodies and neurites at 1 through 15 DIV. The immunoreactivity for smg p25A or synaptophysin was not observed in astrocytes and this finding was confirmed by an immunoblot analysis. These results indicate that smg p25A as well as synaptophysin is present exclusively in neurons and suggest that these two synapse-associated proteins have different sites of function and different kinetics of synthesis, transport, and/or turnover in cultured hippocampal neurons.

Effects of dexamethasone on the expression of myelin basic protein, proteolipid protein, and glial fibrillary acidic protein genes in developing rat brain.

Effects of dexamethasone (DEX) on the relative abundance of myelin basic protein (MBP), proteolipid protein (PLP) and glial fibrillary acidic protein (GFAP) mRNAs in the developing rat brain were examined. After DEX (1.0 mg/kg body weight) or saline was administered intraperitoneally to 3-day-old rats for 7 consecutive days, wet weight, DNA content and the relative abundance of the glia-specific mRNAs in cerebrum and cerebellum were analyzed at postnatal days (P) 10, 20 and 30. DEX decreased both wet weight and DNA content in cerebellum more profoundly than in cerebrum. The appearance of MBP, PLP and GFAP mRNAs in cerebellum preceded that in cerebrum in the control group. In cerebrum, the relative abundance of MBP and PLP mRNAs was significantly less in the DEX group than that in the control group at P20 and P30. The relative abundance of the GFAP mRNA was significantly less in the DEX group than in the control group at P10 and P20, but there was no significant difference at P30. In cerebellum, a significant decrease in the abundance of MBP, PLP and GFAP mRNAs in the DEX group was observed only at P10 but not at P20 and P30. Our findings indicate that DEX suppresses expression of genes related to glial functions, especially myelination when administered in the early postnatal period.

Effect of dexamethasone on pulmonary surfactant metabolism in hyperoxia-treated rat lungs.

We have examined the effect of dexamethasone on the metabolism of pulmonary surfactant in normal and hyperoxia-treated rats. The relative abundance of the surfactant-specific apoprotein A (SP-A) mRNA in lung tissues and the contents of disaturated phosphatidylcholine (DSPC) and SP-A were measured in bronchoalveolar lavage fluids and in lung tissues in 4-wk-old rats exposed to room air or greater than 90% oxygen for 7 d with or without simultaneous treatment with dexamethasone (0.5 mg/kg body wt for 7 d). The relative abundance of the SP-A mRNA was marginally increased by hyperoxia (1.3-fold over controls). Dexamethasone increased the relative abundance of the SP-A mRNA to a level comparable to that with hyperoxia treatment (1.5-fold over controls). In lavage fluids, the contents of DSPC and SP-A were increased by 4- and 6-fold over controls by hyperoxia, respectively, but they were increased only by 2-fold by dexamethasone. In lung tissues, the contents of DSPC and SP-A were increased by 3- and 2-fold over controls by hyperoxia, respectively. These values in lung tissues in the air-exposed rats were not significantly increased by dexamethasone. In hyperoxia-treated rats, dexamethasone did not significantly affect the relative abundance of the SP-A mRNA level and the contents of DSPC and SP-A in lavage fluids and lung tissues. These results indicate that mechanisms other than increased synthesis of SP-A are involved in hyperoxia-induced SP-A accumulation and that dexamethasone does not affect the abnormal accumulation of pulmonary surfactant induced by hyperoxia.

Differential expression of the N-myc, c-fos, and smg p25A genes in human neuroblastoma cells during neuronal and Schwannian differentiation.

We have examined expression of the N-myc, c-fos and smg p25A genes in two human neuroblastoma cell lines during their differentiation. The decrease in the N-myc gene expression and the increase in the c-fos gene expression are observed during the differentiation of NB-1 cells into neuronal cells and of GOTO cells into Schwann-type cells. On the other hand, the smg p25A, a ras p21-like small GTP-binding protein, gene expression is increased in NB-1 cells but not in GOTO cells during their differentiation, suggesting that smg p25A is closely associated with the neuronal phenotype of neuroblastoma cells.

Expression of smg p25A, a ras p21-like small GTP-binding protein, during postnatal development of rat cerebellum.

Changes in expression and localization of smg p25A, a ras p21-like small GTP-binding protein, in developing rat brain were analyzed in comparison with those of synaptophysin, a well-known synaptic vesicle-specific protein. The smg 25A mRNA was detected in whole brain of rat fetus at 14 days of gestation and its level was increased along with the age and reached the maximum level at postnatal day (P) 20. In postnatal cerebellum, the smg25A mRNA level was also increased age-dependently and the maximum level was observed at P30. Immunoblot analysis with an anti-smg p25A monoclonal antibody (MAb SG-11-7) and an anti-synaptophysin monoclonal antibody (SY 38) showed that expression of both smg p25A and synaptophysin was increased age-dependently in postnatal rat cerebellum. By immunofluorescent cytochemical study with the anti-smg p25A antibody, bright fluorescence was observed in the molecular layer of cerebellum and it was increased in accordance with the cerebellar development. In early postnatal cerebellum, the perikarya of Purkinje cells and the white matter were brightly stained with the antibody, but the fluorescence of these portions was faint in adult cerebellum. The anti-synaptophysin monoclonal antibody also stained the molecular layer of cerebellum but the perikarya of Purkinje cells and the white matter had only a weak immunoreactivity with the antibody irrespective of the age. These results indicate that smg p25A is predominantly present in the nerve terminals and that its amount is increased along with the development of postnatal rat cerebellum. Our results also suggest that smg p25A and synaptophysin have the different kinetics of synthesis, transport, and/or turnover in developing rat cerebellum.