Ex Vivo Modeling of Multidomain Peptide Hydrogels with Intact Dental Pulp.

Preservation of a vital dental pulp is a central goal of restorative dentistry. Currently, there is significant interest in the development of tissue engineering scaffolds that can serve as biocompatible and bioactive pulp-capping materials, driving dentin bridge formation without causing cytotoxic effects. Our earlier in vitro studies described the biocompatibility of multidomain peptide (MDP) hydrogel scaffolds with dental pulp-derived cells but were limited in their ability to model contact with intact 3-dimensional pulp tissues. Here, we utilize an established ex vivo mandible organ culture model to model these complex interactions. MDP hydrogel scaffolds were injected either at the interface of the odontoblasts and the dentin or into the pulp core of mandible slices and subsequently cultured for up to 10 d. Histology reveals minimal disruption of tissue architecture adjacent to MDP scaffolds injected into the pulp core or odontoblast space. Additionally, the odontoblast layer is structurally preserved in apposition to the MDP scaffold, despite being separated from the dentin. Alizarin red staining suggests mineralization at the periphery of MDP scaffolds injected into the odontoblast space. Immunohistochemistry reveals deposition of dentin sialophosphoprotein by odontoblasts into the adjacent MDP hydrogel, indicating continued functionality. In contrast, no mineralization or dentin sialophosphoprotein deposition is evident around MDP scaffolds injected into the pulp core. Collagen III expression is seen in apposition to gels at all experimental time points. Matrix metalloproteinase 2 expression is observed associated with centrally injected MDP scaffolds at early time points, indicating proteolytic digestion of scaffolds. Thus, MDP scaffolds delivered centrally and peripherally within whole dental pulp tissue are shown to be biocompatible, preserving local tissue architecture. Additionally, odontoblast function and pulp vitality are sustained when MDP scaffolds are intercalated between dentin and the odontoblast region, a finding that has significant implications when considering these materials as pulp-capping agents.

Histone deactylase inhibition combined with behavioral therapy enhances learning and memory following traumatic brain injury.

Traumatic brain injury (TBI) induces a number of pathological events ranging from neuronal degeneration and tissue loss to impaired neuronal plasticity and neurochemical dysregulation. In rodents, exposure of brain-injured animals to environmental enrichment has been shown to be an effective means of enhancing learning and memory post-injury. Recently, it has been discovered that environmental enrichment may enhance neuronal plasticity through epigenetic changes that involve enhanced histone acetylation, a property that can be mimicked by the use of histone deactylase (HDAC) inhibitors. We therefore evaluated the consequences of the HDAC inhibitor sodium butyrate on the learning and memory of brain-injured mice. In contrast to a previous report using a mouse neurodegeneration model, sodium butyrate (1.2 g/kg daily for four weeks) did not improve learning and memory when tested after the completion of the drug treatment paradigm. In addition, sodium butyrate administration during the reported period of neurodegeneration (days 0-5) also offered no benefit. However, when administered concurrently with training in the Morris water maze task (beginning on day 14 post-injury), sodium butyrate improved learning and memory in brain-injured mice. Interestingly, when these mice were subsequently tested in an associative fear conditioning task, an improvement was observed. Taken together, our findings indicate that HDAC inhibition may mimic some of the cognitive improvements seen following enriched environment exposure, and that the improvement is observed when the treatment is carried out current with behavioral testing.

Persistent working memory dysfunction following traumatic brain injury: evidence for a time-dependent mechanism.

The prefrontal cortex is highly vulnerable to traumatic brain injury (TBI) resulting in the dysfunction of many high-level cognitive and executive functions such as planning, information processing speed, language, memory, attention, and perception. All of these processes require some degree of working memory. Interestingly, in many cases, post-injury working memory deficits can arise in the absence of overt damage to the prefrontal cortex. Recently, excess GABA-mediated inhibition of prefrontal neuronal activity has been identified as a contributor to working memory dysfunction within the first month following cortical impact injury of rats. However, it has not been examined if these working memory deficits persist, and if so, whether they remain amenable to treatment by GABA antagonism. Our findings show that working memory dysfunction, assessed using both the delay match-to-place and delayed alternation T-maze tasks, following lateral cortical impact injury persists for at least 16 weeks post-injury. These deficits were found to be no longer the direct result of excess GABA-mediated inhibition of medial prefrontal cortex neuronal activity. Golgi staining of prelimbic pyramidal neurons revealed that TBI causes a significant shortening of layers V/VI basal dendrite arbors by 4 months post-injury, as well as an increase in the density of both basal and apical spines in these neurons. These changes were not observed in animals 14 days post-injury, a time point at which administration of GABA receptor antagonists improves working memory function. Taken together, the present findings, along with previously published reports, suggest that temporal considerations must be taken into account when designing mechanism-based therapies to improve working memory function in TBI patients.

Performance in long-term memory tasks is augmented by a phosphorylated growth factor receptor fragment.

To elucidate the role of enhanced phosphoinositide-3-kinase (PI3-kinase) activity in memory, a synthetic phosphopeptide (TAT-YPMDM) containing the p85 regulatory subunit receptor-binding motif (YXXM) coupled to the cell transduction domain of HIV-TAT protein was employed. This phosphopeptide bound the p85 subunit of PI3-kinase, and was internalized by both granule and pyramidal neurons when injected into the hippocampus. Increased lipid kinase activity and enhanced phosphorylation of the PI3-kinase substrates Akt (protein kinase B) and ribosomal S6 kinase were associated with TAT-YPMDM administration. Bilateral infusion of the phosphopeptide into the dorsal hippocampus after training improved performance in three hippocampus-dependent memory tasks: contextual fear conditioning, trace fear conditioning, and the Morris water maze. Both the biochemical and behavioral effects of the TAT-YPMDM phosphopeptide could be blocked by wortmannin. No effect was observed when a nonphosphorylated peptide (TAT-YMDM), or a second, unrelated phosphopeptide (TAT-YPLDL) was utilized. In addition, infusion of the TAT-YPMDM phosphopeptide did not interfere with memory acquisition or 4 hr memory. In addition, pretesting administration did not affect the ability to recall a previously established long-term memory. These findings suggest that stimulation of PI3-kinase activity by phosphorylated receptor fragments containing the YMDM motif augments long-term memory.

Expression of cytochromes P450 4F4 and 4F5 in infection and injury models of inflammation.

Lipopolysaccharide (LPS) treatment of rats suppresses CYP 4F4 and 4F5 expression by 50 and 40%, respectively, in a direct fashion occurring in the liver. This contention is borne out by essentially parallel dose-dependent changes observed upon treatment of rat hepatocyte cultures with LPS. An alternate avenue of triggering the inflammatory cascade is traumatic brain injury by controlled cortical impact. Such injury brings about a dramatic change in the expression of CYP 4F4 and 4F5 mRNA which reaches its greatest effect 24 h after impact compared with sham-operated but uninjured controls. At time points after 24 h the expression of both isoforms increases dramatically reaching highest levels at 2 weeks post-injury. These changes in mRNA expression are mirrored by changes in protein expression. The results are consistent with the notion that immediately after injury concentrations of leukotriene and prostaglandin mediators are elevated by decreased CYP 4F concentrations. As time after injury increases those conditions reverse. Increased CYP 4F expression leads to diminished concentrations of leukotriene and prostaglandin mediators and then to recovery and repair.

The role of extracellular signal-regulated kinase in cognitive and motor deficits following experimental traumatic brain injury.

Traumatic brain injury (TBI) causes neuronal death and alters the plasticity (e.g. morphology) of surviving neurons. Both of these events contribute to TBI-associated neurological deficits, such as memory dysfunction. Although a majority of current research is directed towards identifying biochemical cascades responsible for cell death, little is known about mechanisms of altered neuronal plasticity following TBI. Extracellular signal-regulated kinases (Erk1 and 2) play a critical role in growth and have been implicated in long-lasting neuronal plasticity and memory storage. The activation of Erk following TBI was investigated utilizing an antibody that specifically binds to dually phosphorylated Erk. Using this antibody, we report that lateral cortical impact injury in rats increases Erk phosphorylation both in the cortex and the hippocampus as early as 10 min post-injury. Double immunostaining experiments using either a neuron-specific or an astroglial-specific marker show that the active Erk is localized almost exclusively in neuronal cells. Furthermore, the increase in phospho-Erk immunoreactivity was initially localized to axons and at later time points was observed to be predominantly in the cell soma. This suggests that Erk redistributed over time and may play a role in retrograde signaling. Administration of inhibitors of the Erk cascade worsened retrograde amnesia, impaired performances in hippocampus- and amygdala-dependent memory tasks, and exacerbated motor deficits following TBI. Furthermore, inhibition of this cascade did not have any overt effects on cell survival, but altered neuronal morphology as detected by a dendritic-specific marker. These findings suggest that the Erk cascade plays an essential role for the maintenance of neuronal function and plasticity following TBI.

Enhanced neurogenesis in the rodent hippocampus following traumatic brain injury.

Recent studies have shown that neurogenesis in the dentate gyrus of the rodent hippocampus continues throughout life. Several physiological and pathological conditions have been reported to alter the rate of progenitor cell division resulting in the increased production of mature granule neurons. Excitotoxic and mechanical lesions of the granule cell layer also stimulate the proliferation of precursor cells suggesting that the death of pre-existing granule neurons may act as a trigger for enhanced neurogenesis. Hippocampal pyramidal neurons, and to a lesser extent granule neurons, have been reported to die as a result of traumatic brain injury in rodents. To determine if the proliferation of precursor cells is enhanced as a result of brain injury in rodents, newly divided cells were labeled with the thymidine analog, bromodeoxyuridine (BrdU). Traumatic brain injury increased the production of BrdU-labeled cells in the dentate gyrus with a maximal rate observed at 3 days post-injury. These cells, a proportion of which co-localize with the immature neuronal marker TOAD-64, implanted themselves into the granule cell layer where they accumulated over time. When examined 1 month post-injury, the majority of BrdU-labeled cells co-labeled with the mature neuronal marker calbindin. These findings show that traumatic brain injury increases neurogenesis in the granule cell layer and suggests that these new cells may contribute to the function of the hippocampus.

The effects of socioeconomic status and increased body mass index on cardiovascular disease in African-American women.

Today within the United States, CVD is the leading cause of death in women with the highest mortality being seen in African-American women. A review of literature revealed that over the last decade, within the United States, there has been an overall reduction in the death rate due to CVD. However, the rate of decline has been less for women than for men and less for African-American women than for White women. Findings from some studies indicate that African-American women have increased risk factors as compared to other ethnic groups for CVD based upon conditions and behaviors affecting lifestyle. Fortunately, most of the CVD risk factors are modifiable and their occurrence can be widely prevented. Therefore, it is imperative that health care providers approach the issue of risk factors for CVD in African-American women as a heart disease epidemic. This approach is necessary if the United States is going to improve the health of all Americans, eliminate disparities, and improve the quality of life.

Caspase activity plays an essential role in long-term memory.

Activation of intracellular second messenger cascades has been linked to learning and memory in various organisms. Identification of down-stream targets of these second messengers that play a role in learning and memory is an active area of research. Recently, it has been reported that increases in intracellular calcium can activate a cysteine-dependent aspartate-directed protease (caspase) cascade in mice. Using an antibody that selectively recognizes activated caspase-3, we detected the presence of this enzyme in hippocampal neurons. Inhibition of caspase activity in the hippocampus blocked long-term, but not short-term, spatial memory. These results suggest that a caspase-mediated cellular event(s) in hippocampal neurons is critical for long-term spatial memory storage.

Regional expression and role of cyclooxygenase-2 following experimental traumatic brain injury.

Prostaglandins, potent mediators of inflammation, are generated from arachidonic acid (AA) via the action of cyclooxygenase-1 and -2 (COX-1 and COX-2). In this study, we report that lateral cortical impact injury in rats significantly increases COX-2 protein levels both in the cortex surrounding the injury site and the ipsilateral hippocampus. COX-2 protein level was elevated as early as 3 h postinjury and persisted for up to 3 days. Increases in immunoreactivity were detected not only in the adjacent cortex and hippocampus, but were also observed in the contralateral cortex and hippocampus, the ipsilateral piriform cortex and the ipsilateral amygdaloid complex. COX-2 immunoreactive cells appear morphologically normal and do not present any of the characteristic features of apoptosis. Double immunostaining experiments using either a neuron-specific or an astroglial-specific marker show that the expression of COX-2 is localized almost exclusively in neuronal cells. Administration of the COX-2 inhibitor 4-[5-(4-methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfona mide (celecoxib, marketed as Celebrex) worsens motor, but not cognitive, performance, suggesting that COX-2 induction following traumatic brain injury may play a protective role.

Requirement for DARPP-32 in progesterone-facilitated sexual receptivity in female rats and mice.

DARPP-32, a dopamine- and adenosine 3',5'-monophosphate (cAMP)-regulated phosphoprotein (32 kilodaltons in size), is an obligate intermediate in progesterone (P)-facilitated sexual receptivity in female rats and mice. The facilitative effect of P on sexual receptivity in female rats was blocked by antisense oligonucleotides to DARPP-32. Homozygous mice carrying a null mutation for the DARPP-32 gene exhibited minimal levels of P-facilitated sexual receptivity when compared to their wild-type littermates. P significantly increased hypothalamic cAMP levels and cAMP-dependent protein kinase activity. These increases were not inhibited by a D1 subclass dopamine receptor antagonist. P also enhanced phosphorylation of DARPP-32 on threonine 34 in the hypothalamus of mice. DARPP-32 activation is thus an obligatory step in progestin receptor regulation of sexual receptivity in rats and mice.

Inhibitors of tripeptidyl peptidase II. 2. Generation of the first novel lead inhibitor of cholecystokinin-8-inactivating peptidase: a strategy for the design of peptidase inhibitors.

The cholecystokinin-8 (CCK-8)-inactivating peptidase is a serine peptidase which has been shown to be a membrane-bound isoform of tripeptidyl peptidase II (EC 3.4.14.10). It cleaves the neurotransmitter CCK-8 sulfate at the Met-Gly bond to give Asp-Tyr(SO(3)H)-Met-OH + Gly-Trp-Met-Asp-Phe-NH(2). In seeking a reversible inhibitor of this peptidase, the enzymatic binding subsites were characterized using a fluorimetric assay based on the hydrolysis of the artificial substrate Ala-Ala-Phe-amidomethylcoumarin. A series of di- and tripeptides having various alkyl or aryl side chains was studied to determine the accessible volume for binding and to probe the potential for hydrophobic interactions. From this initial study the tripeptides Ile-Pro-Ile-OH (K(i) = 1 microM) and Ala-Pro-Ala-OH (K(i) = 3 microM) and dipeptide amide Val-Nvl-NHBu (K(i) = 3 microM) emerged as leads. Comparison of these structures led to the synthesis of Val-Pro-NHBu (K(i) = 0.57 microM) which served for later optimization in the design of butabindide, a potent reversible competitive and selective inhibitor of the CCK-8-inactivating peptidase. The strategy for this work is explicitly described since it illustrates a possible general approach for peptidase inhibitor design.

Sphingosine-1-phosphate induces apoptosis of cultured hippocampal neurons that requires protein phosphatases and activator protein-1 complexes.

In this study, we report that mobilization of internal Ca2+ by sphingosine-1-phosphate, a metabolite of ceramide, induces apoptosis in cultured hippocampal neurons. This sphingosine-1-phosphate-induced apoptosis is dependent upon the activation of protein phosphatases, possibly calcineurin and phosphatase 2A (or a related phosphatase). In addition, pretreatment of neurons with double-stranded oligonucleotides containing the metallothionein phorbol-12-myristate-13-acetate response element sequence as transcription factor decoys suppressed apoptosis. In contrast, double-stranded oligonucleotides containing either the c-jun or SV40 phorbol-12-myristate-13-acetate response element sequences were ineffective. Electrophoretic mobility shift assays and supershift assays revealed that c-Fos-containing activator protein- complexes preferentially bound the metallothionein phorbol-12-myristate-13-acetate response element sequence-containing oligonucleotides. Furthermore, antisense oligonucleotides to c-fos and c-jun were also protective. The apoptotic death of hippocampal neurons has been hypothesized to contribute to the cognitive impairments observed following insults to the brain. While increases in intracellular calcium are thought to be key mediators of neuronal apoptosis, the biochemical cascade(s) activated as a result of increased Ca2+ which mediates apoptosis of hippocampal neurons is (are) not well understood. The findings presented in this study suggest that mobilization of internal calcium via prolonged exposure of sphingosine-1-phosphate induces apoptosis of hippocampal neurons in culture. Sustained increases in intracellular calcium activate a phosphatase cascade that includes calcineurin and a phosphatase 2A-like phosphatase, and leads to the expression of genes containing metallothionein phorbol-12-myristate-13-acetate response element (TGAGTCA)-type enhancer sequences. The expression of genes containing TGAGTCA-type enhancer sequences appears to be essential for sphingosine-1-phosphate-induced apoptosis of hippocampal neurons.

A mitogen-activated protein kinase cascade in the CA1/CA2 subfield of the dorsal hippocampus is essential for long-term spatial memory.

Behavioral, biophysical, and pharmacological studies have implicated the hippocampus in the formation and storage of spatial memory. However, the molecular mechanisms underlying long-term spatial memory are poorly understood. In this study, we show that mitogen-activated protein kinase (MAPK, also called ERK) is activated in the dorsal, but not the ventral, hippocampus of rats after training in a spatial memory task, the Morris water maze. The activation was expressed as enhanced phosphorylation of MAPK in the pyramidal neurons of the CA1/CA2 subfield. In contrast, no increase in the percentage of phospho-MAPK-positive cells was detected in either the CA3 subfield or the dentate gyrus. The enhanced phosphorylation was observed only after multiple training trials but not after a single trial or after multiple trials in which the location of the target platform was randomly changed between each trial. Inhibition of the MAPK/ERK cascade in dorsal hippocampi did not impair acquisition, but blocked the formation of long-term spatial memory. In contrast, intrahippocampal infusion of SB203580, a specific inhibitor of the stress-activated MAPK (p38 MAPK), did not interfere with memory storage. These results demonstrate a MAPK-mediated cellular event in the CA1/CA2 subfields of the dorsal hippocampus that is critical for long-term spatial memory.

Sequestration of cAMP response element-binding proteins by transcription factor decoys causes collateral elaboration of regenerating Aplysia motor neuron axons.

Axonal injury increases intracellular Ca2+ and cAMP and has been shown to induce gene expression, which is thought to be a key event for regeneration. Increases in intracellular Ca2+ and/or cAMP can alter gene expression via activation of a family of transcription factors that bind to and modulate the expression of CRE (Ca2+/cAMP response element) sequence-containing genes. We have used Aplysia motor neurons to examine the role of CRE-binding proteins in axonal regeneration after injury. We report that axonal injury increases the binding of proteins to a CRE sequence-containing probe. In addition, Western blot analysis revealed that the level of ApCREB2, a CRE sequence-binding repressor, was enhanced as a result of axonal injury. The sequestration of CRE-binding proteins by microinjection of CRE sequence-containing plasmids enhanced axon collateral formation (both number and length) as compared with control plasmid injections. These findings show that Ca2+/cAMP-mediated gene expression via CRE-binding transcription factors participates in the regeneration of motor neuron axons.

alpha-Tocopheryl quinone is converted into vitamin E in man.

The conversion of alpha-tocopheryl quinone into alpha-tocopherol in humans has been demonstrated. A male subject was given an oral dose of 400 mg of alpha-3,5-[(C2H3)2]-tocopheryl quinone with an evening meal. Analysis of plasma 15 h later by lipid extraction and subsequent GC-MS single ion monitoring revealed the presence of alpha-[5,7-(C2H3)2]-tocopherol at a concentration of 0.4 microM, representing 0.8% of the total tocopherol in the plasma sample. This experiment clearly demonstrates that orally administered alpha-tocopheryl quinone is converted in a low overall yield to alpha-tocopherol in humans. The conversion to alpha-tocopherol of that portion of the quinone dose which was actually absorbed into the blood stream may, however, have been fairly efficient.

Characterization and phosphorylation of CREB-like proteins in Aplysia central nervous system.

Studies in Aplysia californica indicate that cAMP-mediated gene expression is necessary for long-term facilitation, a correlate of long-term memory. It has been shown that blocking the expression of cAMP-inducible genes in sensory neurons impedes long-term facilitation without any effect on short-term facilitation. Specifically, blocking the binding of CREB-like proteins or inhibiting the expression of a cAMP-inducible gene, C[symbon: see text]EBP, impairs long-term facilitation. In this report, we show the presence of a family of CREB-like proteins in Aplysia CNS that specifically bind to the CRE sequence and cross-react with rat CREB antibodies. Similar to mammalian CREB proteins, Aplysia homologues interact with each other via leucine zipper domains. This interaction can be disrupted by peptides containing the CREB leucine zipper sequence. We demonstrate that a 43 kDa CREB-like protein present in CNS extracts can be phosphorylated in vitro by cAMP-dependent protein kinase A. Moreover, exposure of ganglia to serotonin (5-HT), a transmitter involved in long-term facilitation, increases the phosphorylation of this protein. This biochemical data further supports the involvement of CREB-like proteins in memory storage.

Neuronal activity increases the phosphorylation of the transcription factor cAMP response element-binding protein (CREB) in rat hippocampus and cortex.

Activity-mediated gene expression is thought to play an important role in many forms of neuronal plasticities. We have used pentylenetetrazol-induced seizure that produces synchronous and sustained neuronal activity as a model to examine the mechanism(s) of gene activation. The transcription factor CREB (Ca2+/cAMP response element-binding protein) is thought to be necessary for long-term memory formation both in invertebrates and vertebrates. When phosphorylated on Ser133 either by cAMP-dependent protein kinase and/or Ca2+/calmodulin-dependent protein kinases, CREB increases transcription of genes containing the CRE (cAMP response element) sequence. Using an antibody that detects Ser133-phosphorylated CREB protein, we show that CREB phosphorylation is maximal between 3 and 8 min after the onset of seizure activity and declines slowly both in the hippocampus and the cortex. The total amount of CREB protein did not change at the time points examined. The increased phosphorylation of CREB protein is preceded by an increase in the amount of cAMP, suggestive of cAMP-dependent protein kinase activation, in the hippocampus and activation of Ca2+/calmodulin-dependent protein kinases in the cortex. Subsequent to CREB phosphorylation, the expression of the CRE-containing gene, c-fos, and the AP-1 complexes (heterodimers of Fos and Jun family members) is increased. These findings support the role of CREB-mediated gene expression in activity-dependent neuronal plasticities.

Characterization and inhibition of a cholecystokinin-inactivating serine peptidase.

A cholecystokinin (CCK)-inactivating peptidase was purified and identified as a membrane-bound isoform of tripeptidyl peptidase II (EC 3.4.14.10), a cytosolic subtilisin-like peptidase of previously unknown functions. The peptidase was found in neurons responding to cholecystokinin, as well as in non-neuronal cells. Butabindide, a potent and specific inhibitor, was designed and shown to protect endogenous cholecystokinin from inactivation and to display pro-satiating effects mediated by the CCKA receptor.

Deferoxamine improves spatial memory performance following experimental brain injury in rats.

Traumatic brain injury (TBI) causes impairments of both motor and spatial memory performances. Research is only beginning to reveal the biochemical mechanism(s) underlying these deficits. It has been postulated that reactive oxygen species such as the superoxide and hydroxyl radicals, as well as the peroxynitrite anion, are generated by injury and may play a critical role in the observed memory deficits. The highly reactive hydroxyl radical, which is thought to contribute to neuronal toxicity, can be generated by an iron-catalyzed reaction. The source of this iron (or iron-bound proteins) could be a compromise of the blood-brain barrier, which can occur following TBI. In this report, we investigate the ability of deferoxamine, a scavenger of free iron, the hydroxyl radical and the peroxynitrite anion, to facilitate behavioral recovery following a controlled cortical impact of rats. Intraperitoneal administration of this drug prior to the injury did not affect the rate of recovery from motor deficits in comparison to vehicle (saline)-injected animals. However, deferoxamine-treated animals showed significant improvement in spatial memory performance in a Morris water maze task. Volumetric analysis of cortical tissue loss showed no significant differences between vehicle- and drug-injected animals. Similarly, histological examination of the hippocampus did not reveal any gross differences between the two groups. These results indicate that deferoxamine improves spatial memory performance, possibly through protection from neuronal dysfunction.