Identification of Regulatory Elements Necessary for the Expression of the COL1A1 Promoter in Murine Odontoblasts

Recent studies have indicated that odontoblasts and osteoblasts have unique regulatory mechanisms that control COL1A1 gene expression. We are currently examining the regulation of COL1A1 gene expression in odontoblasts and have produced transgenic mice containing various collagen promoter constructs fused to the indicator gene, chloramphenicol acetyl transferase (CAT). Mandibular first molars were removed from jaws of transgenic mice. Some teeth were assayed for CAT activity (CAT diffusion assays), others were fixed and prepared for immunohistochemistry (CAT antibodies).

Our results indicate the CAT activity was present in tooth germs containing promoter constructs longer than 1.719 kb. Immunoreactivity to CAT was confined to the odontoblast cell layer. No CAT activity was present in tooth germs containing a 1.670 kb construct.

These data suggest that there are important regulatory elements located between -1.719 kb and -1.670 kb on the collagen promoter in odontoblasts. Examination of sequences in this region of the promoter demonstrates consensus with those known to be involved with binding of translation products of homeobox genes.     

Dentin Mineralization and the Role of Odontoblasts in Calcium Transport

Dentin is formed by two simultaneous processes, in which the odontoblasts are instrumental—the formation of the collagenous matrix, and mineral crystal formation in this matrix. This pattern of formation is similar to that of bone, another mineralized connective tissue. Dentin and bone also have chemical compositions which are similar but with distinct differences. It is of fundamental importance to understand how the ions constituting the inorganic phase are transported from the circulation to the site of mineral formation and how this transport is regulated. For dentinogenesis, calcium is essentially the only ion for which data are available. Recent evidence suggests that a major portion of the Ca²⁺ ions are transported by a transcellular route, thus being under cellular control. The cells maintain a delicate Ca²⁺ ion balance by the concerted action of transmembraneous transport mechanisms, including Ca-ATPase, Na⁺/Ca²⁺ exchangers and calcium channels, and of intracellular Ca²⁺-binding proteins. The net effect of this is a maintenance of a sub-micromolar intracellular Ca²⁺ activity, and an extracellular accumulation of Ca²⁺ ions in predentin, at the mineralization front. Predentin can be regarded as a zone of formation and maturation of the scaffolding collagen web of the dentin organic matrix. In addition to collagen, it contains little but proteoglycan. Simultaneous with mineral formation, additional non-collagenous macromolecules are added to the extracellular matrix of dentin, these presumably being transported within the odontoblast process. Among these are highly phosphorylated dentin phosphoprotein (phospho-phoryn) and another pool of proteoglycan. The functionality of this may be explained by the fact that polyanionic macromolecules are capable of inducing the formation of hydroxyapatite at ionic conditions resembling those in vivo. They can also inhibit mineral growth and regulate crystal size.     

Vesicular Glutamate Transporter Immunoreactivity in the Periodontal Ligament of the Rat Incisor

The distribution of three vesicular glutamate transporter (VGluT) isoforms, VGluT1, VGluT2, and VGluT3, were investigated in the trigeminal ganglion of the periodontal ligament in the rat incisor—a receptive field of trigeminal ganglion neurons. In the trigeminal ganglion, mRNAs for all VGluT isoforms were detected and proteins were observed in the cytoplasm of trigeminal ganglion cells. VGluT1 immunoreactions were localized within the cytoplasm for all sizes of trigeminal neurons, although predominately in medium–large trigeminal neurons. Double‐labeling showed that most VGluT1 contained both VGluT2 and VGluT3. In the periodontal ligament of the incisor, the Ruffini endings, principal periodontal mechanoreceptors, displayed VGluT1 and VGluT2 immunoreactivities. However, lacked immunoreactions for VGluT3. At the electron microscopic level, VGluT1 immunoreactions were localized around the vesicle membranes at the axon terminal of Ruffini endings. The present results indicate that VGluT is expressed in the sensory nerve endings where apparent synapses are not present. Thus, glutamate in the sensory nerve endings is thought to be used in metabotropic functions. This is because glutamate is a general metabolic substrate, and/or acts as a neurotransmitter as proposed in muscle spindles. Anat Rec, 2012. © 2011 Wiley Periodicals, Inc.     

Vesicular release of ATP at central synapses

Adenosine triphosphate (ATP) acts as a fast excitatory transmitter in several regions of the central nervous system (CNS) including the medial habenula, dorsal horn, locus coeruleus, hippocampus, and somatosensory cortex. Postsynaptic actions of ATP are mediated through an extended family of P2X receptors, widely expressed throughout the CNS. ATP is released via several pathways, including exocytosis from presynaptic terminals and diffusion through large transmembrane pores (e.g., hemichannels, P2X₇ receptors, or volume-sensitive chloride channels) expressed in astroglial membranes. In presynaptic terminals, ATP is accumulated and stored in the synaptic vesicles. In different presynaptic terminals, these vesicles may contain ATP only or ATP and another neurotransmitter [e.g., γ-amino-butyric acid (GABA) or glutamate]; in the latter case, two transmitters can be coreleased. Here, we discuss the mechanisms of vesicular release of ATP in the CNS and present our own data, which indicate that in central neuronal terminals, ATP is primarily stored and released from distinct pool of vesicles; the release of ATP is not synchronized either with GABA or with glutamate.     

Acetylcholinesterase Activity,Choline Acetyltransferase and Vesicular Acetylcholine Transporter Immunoreactivities in the Rat Adrenal Gland During Postnatal Development

From postnatl‐day‐0 to postnatal‐day‐2, a few acetylcholinesterase (AChE)‐active and choline acetytransferase (ChAT)‐immunoreactive nerve fibers and relatively numerous vesicular acetylcholine transporter (VAChT)‐immunoreactive puncta were observed in the rat adrenal medulla. Despite relatively numerous clear vesicles in the nerve fibers, the synthesis and hydrolysis of acetylcholine may not be fully activated until postnatal‐day‐2. The number of AChE‐active and ChAT‐immunoreactive nerve fibers dramatically increased and that of VAChT‐immunoreactive puncta gradually increased from postnatal‐day‐3 to postnatal‐week‐1. The synthesis and hydrolysis of acetylcholine may be dramatically activated in the nerve fibers of the medulla until postnatal‐week‐1. From postnatal‐week‐2 to postnatal‐week‐3, the number of AChE‐active and the ChAT‐immunoreactive nerve fibers gradually increased and reached the adult levels. The VAChT‐immunoreactive puncta per unit area was maximum number at postnatal‐week‐2. The synthesis and hydrolysis of acetylcholine in the nerve fibers of the medulla may be completed between postnatal‐week‐2 to postnatal‐week‐3. The diameter of the VAChT‐immunoreactive puncta gradually increased from postnatal‐day‐0 with aging. However, the number of the VAChT‐immunoreactive puncta gradually decreased from postnatal‐week‐2 onwards. In electron‐microscopy, the VAChT‐immunoreactive deposits were seen in clusters of clear vesicles, and the diameter of the nerve fibers and the number of clear vesicles at postnatal‐week‐8 increased compared with those at postnatal‐week‐2. The AChE‐active, ChAT‐immunoreactive, and VAChT‐immunoreactive nerve fibers observed around noradrenaline (NA) cells were denser than those around adrenaline (A) cells in the medulla at postnatal‐week‐8. These suggest that the preferential innervation of NA and A cells may cause the differential secretion NA and A. Anat Rec, 292:371–380, 2009. © 2009 Wiley‐Liss, Inc.     

The Second Messenger Response upon Receptor Binding of Parathyroid Hormone in Rat Odontoblasts

To elucidate the progressive disappearance of 25 kDa amelogenin occurring in a narrow space near the surface of enamel, the alkaline soluble fraction which contained 80% of the total proteins was extracted from a newly formed porcine enamel. When this fraction was incubated with the addition of Ca ions in an in vitro system, the degradation of the coexisting amelogenin and enamelin occurred without activation during the incubation period. Although the fraction contained mainly two kinds of metalloproteinases, 56 kDa and 61 kDa gelatinolytic, and 41 kDa and 46 kDa caseinolytic activities, it was demonstrated on amelogenin enzymography that the caseinolytic one was concerned with the conversion of the 25 kDa amelogenin into the 20 kDa amelogenin. The protein distribution of the newly formed enamel indicated that the metalloproteinases degraded the coexisting enamelin and amelogenin imperfectly. Nevertheless, during the next developing stage they demonstrated their full activities. It is suspected that these activities are regulated by Ca ions, which may be increased by a cascade system.     

Computer Model of Nucleotide Transport in a Realistic Porcine Aortic Trifurcation

Adenosine triphosphate (ATP) is a ubiquitous blood borne agonist which is responsible for the regulation of vascular tone via purinogenic signalling pathways. The present study models the transport of ATP in a realistic porcine aortic trifurcation, which includes multiple branches and bifurcations. The focus of the present study is understanding how pulsatile flow effects mass transfer, observing both mean and transient variations. Unlike in the many idealized models which model transport of low diffusion coefficient species, the realistic geometry leads to very different mass transfer characteristics. These include spiral patterns in the distribution of low concentration fluid. Furthermore, the mean ATP distribution was found to be elevated compared with the steady state; this is attributed to the effects of convective mixing. The results strongly implicate that under certain conditions mass transport in pulsatile flow exhibits different hydrolysis characteristics at the endothelium compared with steady state. Transient variations throughout the the cardiac cycle were found to be small. This small transient response is primarily due to low ATP diffusivity.     

The Serotonin Transporter Expression in Drosophila melanogaster

Abstract: The serotonin transporter is an important regulator of serotonergic signaling. In order to analyze where the Drosophila melanogaster ortholog of the mammalian serotonin transporter (dSERT) is expressed in the nervous system, a dSERT antibody serum was used. Ectopic expression studies and loss of function analysis revealed that the dSERT antibody serum specifically recognizes dSERT. It was shown that in the embryonic nervous system dSERT is expressed in a subset of Engrailed-positive neurons. In the larval brain, dSERT is exclusively expressed in serotonergic neurons, all of which express dSERT. dSERT-positive neurons surround almost all brain neuropiles. In the mushroom body of the adult brain, extrinsic serotonergic neurons expressing dSERT engulf the mushroom body lobes. These neurons show regional differences in dSERT and serotonin expression. At the presynaptic terminals, serotonin release is sterically linked to serotonin reuptake. In contrast to this, there are other areas in serotonergic neurons where dSERT expression and/or function are uncoupled from synaptic neurotransmitter recycling and serotonin release. The localization pattern of dSERT can be employed to further understanding and analysis of serotonergic networks.     

Transporter Molecules influence the Gene Expression in HeLa Cells

Progresses in biology and pharmacology led to highly specific bioactive substances, but their poor bioavailability at the site of action is a result of their physico-chemical properties. Various design approaches for transport carrier molecules facilitating the cellular entry of bioactive substances could help to reach their molecular target in cells and tissues. The transfer efficacy and the subsequent pharmacological effects of the cargo molecules are well investigated, but the investigations of effects of the carrier molecules themselves on the target cells or tissues remain necessary. A special attention should be paid to the differential gene expression, particularly in the interpretation of the data achieved by highly specific active pharmaceutical products. After application of transmembrane transport peptides, particularly the pAnt and also the HIV-1 Tat, cells respond with a conspicuous altered gene expression of at least three genes. The PKN1 gene was induced and two genes (ZCD1 and BSG) were slightly repressed. The genes and the chromosomes are described, the moderate differential gene expression graphed, and the ontology is listed.     

P2X7-related modulation of pathological nociception in rats

Growing evidence supports a role for the immune system in the induction and maintenance of chronic pain. ATP is a key neurotransmitter in this process. Recent studies demonstrate that the glial ATP receptor, P2X7, contributes to the modulation of pathological pain. To further delineate the endogenous mechanisms that are involved in P2X7-related antinociception, we utilized a selective P2X7 receptor antagonist, A-438079, in a series of in vivo and in vitro experiments. Injection of A-438079 (10-300 micromol/kg, i.p.) was anti-allodynic in three different rat models of neuropathic pain and it attenuated formalin-induced nocifensive behaviors. Using in vivo electrophysiology, A-438079 (80 micromol/kg, i.v.) reduced noxious and innocuous evoked activity of different classes of spinal neurons (low threshold, nociceptive specific, wide dynamic range) in neuropathic rats. The effects of A-438079 on evoked firing were diminished or absent in sham rats. Spontaneous activity of all classes of spinal neurons was also significantly reduced by A-438079 in neuropathic but not sham rats. In vitro, A-438079 (1 microM) blocked agonist-induced (2,3-O-(4-benzoylbenzoyl)-ATP, 30 microM) current in non-neuronal cells taken from the vicinity of the dorsal root ganglia. Furthermore, A-438079 dose-dependently (0.3-3 microM) decreased the quantity of the cytokine, interleukin-1beta, released from peripheral macrophages. Thus, ATP, acting through the P2X7 receptor, exerts a wide-ranging influence on spinal neuronal activity following a chronic injury. Antagonism of the P2X7 receptor can in turn modulate central sensitization and produce antinociception in animal models of pathological pain. These effects are likely mediated through immuno-neural interactions that affect the release of endogenous cytokines.     

Evidence of Two Types of Odontoblasts during Dentinogenesis in Elasmobranchs

The fine structure of the odontoblasts in the sting rays, Dasyatis akajei Dasyatidae, and Urolophus aurantiacus Urolophidae, was examined using light and transmission electron microscopy. In the dentinogenesis stage, the odontoblasts have been classified into two types, that is, dark cells and light cells, based on differences in their fine structure. Many dark odontoblasts found along the predentine displayed well-developed organelles with secretory activity around the nuclei. They contained large amounts of expanded rER, widely distributed Golgi apparatus and secretory granules. In contrast, light odontoblasts showed a relatively clear cytoplasm and extended long processes which passed through the predentine and penetrated into the dentine. They contained large numbers of microtubules in the processes and many mitochondria around the nuclei. It is suggested that the light odontoblasts play an important part in material transport to the dentine and/or act as a sensory organ of the tooth. The dark odontoblasts seem to produce the organic matrix of the dentine and to prepare for mineralization in the dentine.     

Synaptic potentials recorded in brain slices and their modification by changes in the level of tissue ATP

Summary Postsynaptic responses to electrical stimulation were recorded in olfactory cortex slices maintained in vitro, and their modification by changes in the level of tissue ATP was examined. A slight decrease in the ATP level caused by lowered oxygen tension markedly affects the synaptic transmission. Thus when ATP is decreased by 20%, the amplitude of the synaptic potential already diminishes by 93%. With inhibitors of oxidative phosphorylation, reduction of ATP and suppression of synaptic potential occur to the same extent as that induced by lowered oxygen tension. In contrast, decrease of ATP caused by the inhibition of glycolytic pathway affects the transmission to a lesser extent. With chlorpromazine and phenobarbitone, synaptic transmission is affected more than is expected from the reduction of ATP. When ATP is reduced by various means, the post-tetanic potentiation and frequency potentiation are enhanced. With the reduced ATP level, the post-tetanic potentiation develops and also declines more rapidly than with the normal level of ATP. Reduction of ATP and of the potential caused by anoxia or cyanide can be recovered when the normal metabolic condition of the slice is restored. However, if slices are incubated anaerobically in a glucosefree medium, an irreversible fall in both ATP and the synaptic potential occurs. In contrast with the decrease, increase of ATP up to about 135% of the normal level does not affect transmission.On leave of absence from Behaviour Research Institute, University of Gunma Medical School, Maebashi, Japan. Supported by a grant of Visiting Scientist Programme (April 1968 to March 1969), Japan Society for the Promotion of Science.     

Characterization of nestin expression in the spinal cord of GFP transgenic mice after peripheral nerve injury

Many studies have shown that activation and increase in the number of astrocytes and microglia in the spinal cord participate in the initiation and maintenance of neuropathic pain, but little attention has been paid to the responses of neural progenitor cells to peripheral nerve injury. Nestin, a class VI intermediate filament protein, is expressed both in neuronal and glial progenitors as well as in their common precursors; and nestin-positive cells appear in the brain and spinal cord following various forms of damage to these regions. To clarify the responses of neural progenitor cells to nerve injury, we applied L5 spinal nerve transection (L5-SNT) to nestin-promoter GFP (pNestin-GFP) transgenic mice to narrow the target to them. While pNestin-GFP expression was strongly retained in the ependyma lining the central canal of the transgenic spinal cord even in adulthood, it was markedly reduced in the dorsal horn during postnatal development by day 7. Increases in pNestin-GFP expression and labeling by the proliferation marker 5-bromodeoxyuridine were broadly found in the dorsal horn of adult mice on day 3 after L5-SNT. On the other hand, the activation and increase in number of microglia and astrocytes are restricted to the superficial layer of the dorsal horn, the central terminal of injured primary afferent fibers. Purinergic P2X agonist α, β-MeATP increased [Ca²⁺]i in nestin-positive cells in the superficial layer ipsilateral to nerve injury and P2 receptor antagonists suramin and pyridoxalphosphate-6-azophenyl-2,4-disulphonic acid (PPADS) blocked the expression and elongation of pNestin-GFP fibers in the slice culture of the spinal cord. These results with pNestin-GFP transgenic mice demonstrate that nestin-positive cells proliferate in the dorsal horn in response to peripheral nerve injury and suggest that ATP may contribute to the expression of nestin and activation of neural progenitor cells after nerve injury.     

阿片δ受体激动剂预处理延迟效应保护大鼠心肌的实验研究

目的探讨以阿片δ受体激动剂能否诱导心脏缺血预处理的延迟保护效应及对大鼠心肌缺血-再灌注损伤的保护效果。方法大鼠随机分4组，A、B组以阿片δ受体激动剂DADLE预处理（2mg／kg），C、D组注射生理盐水，24h后4组都制成离体心脏，低温缺血3h，复灌1h。观测心功能、ATP等。其中B、D组在缺血前以优降糖阻止心肌ATP敏感性钾通道的开放。结果A左室压力变化最大速率恢复率和心肌ATP含量都高于B、C、D组，差异有显著性（P〈0．01 or P〈0．05），而B、C、D组间无明显差异。A组心肌丙二醛含量与CK-MB漏出活性明显低于B、C、D组（P〈0．01 or P〈0．05）。结论阿片δ受体激动剂可以诱导预处理的延迟效应，并减轻大鼠心肌的缺血-再灌注损伤，而ATP敏感性钾通道参与介导其效应的机制。     

Immunohistochemical Analysis of Nucleotide Excision Repair Factors XPA and XPB in Adult Rat Brain

To study the regional and cellular distribution of xeroderma pigmentosum group A and B (XPA and XPB) proteins, two nucleotide excision repair (NER) factors, in the mammalian brain we used immunohistochemistry and triple fluorescent immunostaining combined with confocal microscope scanning in brain slices of adult rat brain, including the cerebral cortex, striatum, substantia nigra compacta, ventral tegmental area, red nucleus, hippocampus, and cerebellum. Both XPA and XPB proteins were mainly expressed in neurons, because the XPA‐ or XPB‐immunopositive cells were only costained with NeuN, a specific neuronal marker, but not with glial fibrillary acidic acid, a specific astrocyte marker, in the striatum. Furthermore, XPA‐ and XPB‐positive staining were observed in the neuronal nuclei. Such subcellular distribution was consistent with the location of the NER in the cells. This study provides the first evidence that NER factors XPA and XPB exist in the nuclei of neurons in the brain, suggesting that the NER may play important roles in the process of DNA repair in adult brain neurons. Anat Rec, 291:775–780, 2008. © 2008 Wiley‐Liss, Inc.     

P2X_4受体在大鼠神经系统的分布

目的　　观察Ｐ２Ｘ４受体在大鼠神经系统的分布。方法　　Ｐ２Ｘ４受体特异性抗体的免疫细胞化学染色。结果　　Ｐ２Ｘ４受体阳性神经元主要分布于嗅球、前扣带回皮质、梨状皮质、内侧隔核、杏仁中央核、海马ＣＡ３区、乳头体上核、脚间核、三叉神经中脑核、三叉神经运动核、孤束核、最后区、穹窿下器、小脑皮质的Ｐｕｒｋｉｎｊｅ细胞、延髓和脊髓后角Ⅰ层和Ⅱ层、背根神经节和三叉神经节。结论　　Ｐ２Ｘ４受体阳性结构广泛分布于大鼠神经系统,为ＡＴＰ发挥作用提供了位点。     

Bioelectrical Impedance May Predict Cell Viability During Ischemia and Reperfusion in Rat Liver

Ischemia and reperfusion (I/R) injury is a major cause of hepatic failure after liver surgery, but no method could monitor or predict it real-time during surgery. We measured bioelectrical impedance (BEI) and cell viability to assess the usefulness of BEI during I/R in rat liver. A 70% partial liver ischemia model was used. BEI was measured at various frequencies. Adenosine triphosphate (ATP) content, and palmitic acid oxidation rate were measured, and histological changes were observed in order to quantify liver cell viability. BEI changed significantly during ischemia at low frequency. In the ischemia group, BEI increased gradually during 60 min of ischemia and had a tendency to plateau thereafter. The ATP content decreased below 20% of the baseline level. In the I/R group, BEI recovered to near baseline level. After 24 hr of reperfusion, the ATP contents decreased to below 50% in 30, 60 and 120 min of ischemia and the palmitic acid metabolic rates decreased to 91%, 78%, and 74%, respectively, compared with normal liver. BEI may be a good tool for monitoring I/R during liver surgery. The liver is relatively tolerant to ischemia, however after reperfusion, liver cells may be damaged depending upon the duration of ischemia.     

新生期孤养对成年大鼠行为与5-羟色胺转运体表达的影响

目的:探讨新生期孤养(neonatal isolation,NI)对成年大鼠行为与5-羟色胺转运体(5-HTT)表达的影响。方法:新生的SD大鼠随机分为正常对照(non-handled,NH)组与新生期孤养组,新生期孤养组在出生后2-14d每天孤养4h,断乳后常规饲养。12w龄时,进行旷场、高架十字迷宫和强迫游泳试验,酶联免疫法检测血浆促肾上腺皮质激素与皮质酮、海马与下丘脑5-HT的含量,原位杂交法观察中缝核5-HTTmRNA的表达。结果:高架十字迷宫试验中新生期孤养组大鼠出入开放臂的总次数与进入开放臂的比率显著减少;强迫游泳试验中新生期孤养组大鼠悬浮时间显著增多(P0.05)。新生期孤养组大鼠血浆促肾上腺皮质激素与皮质酮显著升高,海马中5-HT含量显著减少,中缝核5-HTTmRNA表达显著增强(P0.05或P0.01)。结论:新生期孤养可导致大鼠在成年时出现抑郁和焦虑样行为,可能与下丘脑-垂体-肾上腺轴功能亢进、中枢5-HT功能低下有关。