Quantitative determination of lysophosphatidic acids (LPAs) in human saliva and gingival crevicular fluid (GCF) by LC-MS/MS.

Lysophosphatidic acid (LPA) is a phospholipid mediator that plays multiple cellular functions by acting through G protein-coupled LPA receptors. LPAs are known to be key mediators in inflammation, and several lines of evidence suggest a role for LPAs in inflammatory periodontal diseases. A simple and sensitive liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method has been developed and validated to quantify LPA species (LPA 18:0, LPA 16:0, LPA 18:1 and LPA 20:4) in human saliva and gingival crevicular fluid (GCF). LPA 17:0 was used as an internal standard and the LPA species were extracted from saliva by liquid-liquid extraction using butanol. Chromatography was performed using a Macherey-Nagel NUCLEODUR® C8 Gravity Column (125 mm × 2.0 mm ID) with a mixture of methanol/water: 75/25 (v/v) containing 0.5% formic acid and 5 mM ammonium formate (mobile phase A) and methanol/water: 99/0.5 (v/v) containing 0.5% formic acid and 5mM ammonium formate (mobile phase B) at a flow rate of 0.5 mL/min. LPAs were detected by a linear ion trap-triple quadrupole mass spectrometer with a total run time of 8.5 min. The limit of quantification (LOQ) in saliva was 1 ng/mL for all LPA species and the method was validated over the range of 1-200 ng/mL. The method was validated in GCF over the ranges of 10-500 ng/mL for LPA 18:0 and LPA 16:0, and 5-500 ng/mL for LPA 18:1 and LPA 20:4. This sensitive LC-MS/MS assay was successfully applied to obtain quantitative data of individual LPA levels from control subjects and patients with various periodontal diseases. All four LPA species were consistently elevated in samples obtained from periodontal diseases, which supports a role of LPAs in the pathogenesis of periodontal diseases.

Diabetes mellitus: a review of the literature and dental implications.

Diabetes mellitus consists of a group of disorders, which are characterized by a lack of insulin production or insulin resistance. There can be various oral manifestations of diabetes, such as xerostomia and an increased incidence of dental caries. Recently, it has been suggested that periodontitis be added as the sixth complication of diabetes mellitus. It has been shown that uncontrolled or poorly controlled diabetics have a greater incidence of severe periodontal disease compared with those patients who are well controlled or have no diabetes mellitus. This has been found for both type 1 and type 2 diabetics. In addition, the diabetic patient may be predisposed to periodontal disease based on the production of advanced glycation end products, which bind to receptors on specific cells such as the monocyte. The success of periodontal treatment appears to be dependent on the control exhibited by the diabetic patient. The well-controlled diabetic will respond well to periodontal treatment, while the uncontrolled or poorly controlled will often not respond well or be stable in the long-term. Because of the large number of diabetics in the US population, dental therapists should be aware of the interactions of the patient's diabetic status, the proposed treatment, and the possible treatment outcomes as well as complications.

Lysophosphatidic acid and EGF stimulate mitogenesis in human airway smooth muscle cells.

Enhanced proliferation of airway smooth muscle is thought to contribute to the pathogenesis of asthma and other obstructive airway diseases. Lysophosphatidic acid (LPA) is a simple bioactive lipid mediator that stimulates mitogenesis in fibroblasts and some other cell types. The effects of LPA on mitogenesis of cultured human airway smooth muscle cells were determined by measuring [3H]thymidine incorporation into cellular DNA. LPA induced a concentration-dependent stimulation of [3H]thymidine incorporation of a similar magnitude to that induced by serum, with the effects of 50 microM LPA being similar to those of 5% serum. Stimulation by LPA and by serum was almost completely eliminated in cells exposed to pertussis toxin, indicating involvement of a pertussis toxin-sensitive G protein in mitogenic signaling by these agents. Epidermal growth factor (EGF) induced stimulation of a similar magnitude as that with LPA, but the stimulation by EGF was insensitive to pertussis toxin. LPA and EGF, when added together, exhibited a markedly synergistic stimulation of [3H]thymidine incorporation that was typically 10-fold greater than the stimulation with either agent alone. LPA and EGF also stimulated mitogenesis assessed by cell growth, and again LPA and EGF together exhibited synergism. These results suggest the possibility that stimulation of airway smooth muscle cell proliferation by LPA, either alone or by enhancing effects of other growth factors, could play a role in normal airway remodeling or in the pathological proliferation of smooth muscle in various airway diseases.

Neurotrophins and their receptors in nerve injury and repair.

Cytokines are a heterogenous group of polypeptide mediators that have been associated with activation of numerous functions, including the immune system and inflammatory responses. The cytokine families include, but are not limited to, interleukins (IL-I alpha, IL-I beta, ILIra and IL-2-IL-15), chemokines (IL-8/ NAP-I, NAP-2, MIP-I alpha and beta, MCAF/MCP-1, MGSA and RANTES), tumor necrosis factors (TNF-alpha and TNF-beta), interferons (INF-alpha, beta and gamma), colony stimulating factors (G-CSF, M-CSF, GM-CSF, IL-3 and some of the other ILs), growth factors (EGF, FGF, PDGF, TGF alpha, TGF beta and ECGF), neuropoietins (LIF, CNTF, OM and IL-6), and neurotrophins (BDNF, NGF, NT-3-NT-6 and GDNF). The neurotrophins represent a family of survival and differentiation factors that exert profound effects in the central and peripheral nervous system (PNS). The neurotrophins are currently under investigation as therapeutic agents for the treatment of neurodegenerative disorders and nerve injury either individually or in combination with other trophic factors such as ciliary neurotrophic factor (CNTF) or fibroblast growth factor (FGF). Responsiveness of neurons to a given neurotrophin is governed by the expression of two classes of cell surface receptor. For nerve growth factor (NGF), these are p75NTR (p75) and p140trk (referred to as trk or trkA), which binds both BDNF and neurotrophin (NT)-4/5, and trkC receptor, which binds only NT-3. After binding ligand, the neurotrophin-receptor complex is internalized and retrogradely transported in the axon to the soma. Both receptors undergo ligand-induced dimerization, which activates multiple signal transduction pathways. These include the ras-dependent pathway utilized by trk to mediate neurotrophin effects such as survival and differentiation. Indeed, cellular diversity in the nervous system evolves from the concerted processes of cell proliferation, differentiation, migration, survival, and synapse formation. Neural adhesion and extracellular matrix molecules have been shown to play crucial roles in axonal migration, guidance, and growth cone targeting. Proinflammatory cytokines, released by activated macrophages and monocytes during infection, can act on neural targets that control thermogenesis, behavior, and mood. In addition to induction of fever, cytokines induce other biological functions associated with the acute phase response, including hypophagia and sleep. Cytokine production has been detected within the central nervous system as a result of brain injury, following stab wound to the brain, during viral and bacterial infections (AIDS and meningitis), and in neurodegenerative processes (multiple sclerosis and Alzheimer's disease). Novel cytokine therapies, such as anticytokine antibodies or specific receptor antagonists acting on the cytokine network may provide an optimistic feature for treatment of multiple sclerosis and other diseases in which cytokines have been implicated.

Buffers differentially alter the binding of [3H]rauwolscine and [3H]RX821002 to the alpha-2 adrenergic receptor subtypes.

3H-antagonists are known to bind to the alpha-2A adrenergic receptor with higher affinity in glycylglycine buffer than in Tris buffer. The purpose of this study was to examine the effect of buffers on the binding of antagonists to all four subtypes of the alpha-2 adrenergic receptor. Our approach was to examine the effects of glycylglycine, Tris, sodium phosphate (NaPO4) and potassium phosphate buffers on the binding of [3H]rauwolscine, [3H]RX821002, prazosin and oxymetazoline. We found that the affinities for the different subtypes varied with the buffer and the ligands used. Although the Bmax values varied somewhat with the buffers, they were similar for both radioligands for a specific subtype. The highest affinities and Bmax values for both radioligands were generally obtained with NaPO4 buffer. The affinities of antagonists in Tris buffer were always significantly lower than in either NaPO4 or glycylglycine buffer, and the affinities decreased as the concentration of Tris increased. In contrast, the affinity of norepinephrine for the alpha-2B subtype was higher in Tris than in NaPO4 buffer. The buffer effects did not appear to be dependent on the cell membrane composition. There appeared to be some species differences in the effects of buffers on the alpha-2C subtype. These results indicate that buffers affect the binding of antagonists to alpha-2 adrenergic receptors, that not all subtypes are altered in the same manner and that buffers alter the binding of different antagonists differently. It is generally recommended that NaPO4 buffer be used, and that Tris be avoided, when measuring the binding of antagonists to the alpha-2 adrenergic receptor.

Regulation of hamster alpha 1B-adrenoceptors expressed in Chinese hamster ovary cells.

Chinese hamster ovary (CHO) cells were stably transfected to express the hamster alpha 1B-adrenoeceptor, and the function and agonist-induced regulation of the binding properties of these receptors were characterized. The cells expressed approximately 230,000 receptors per cell, with a KD for [3H]prazosin of 140 pM. In assays of competition by epinephrine for [3H]prazosin binding to receptors on intact cells, 88% of the receptors were in a low affinity form. The protein kinase C activator phorbol 12-myristate, 13-acetate (PMA) did not further increase the fraction in the low affinity form, but the protein kinase C inhibitor staurosporine reduced the low affinity fraction to 51%. In sucrose density gradient centrifugation assays of receptor internalization, the percentage of receptors in the light vesicle fraction was 25% for control cells, 53% for epinephrine-pretreated cells, 44% for PMA-pretreated cells, and 53% for cells pretreated with epinephrine plus PMA. Staurosporine completely blocked PMA-induced internalization, but only partially inhibited epinephrine-induced internalization. These results suggest a relationship between low affinity binding and internalization for alpha 1B-adrenoceptors and the involvement of protein kinase C in both processes. Longer-term (24 h) exposure of cells to epinephrine induced an unexpected up-regulation of receptor density of approximately 2-fold that was accompanied by an increase in maximal agonist-stimulated phosphoinositide turnover. These studies document several regulatory differences between alpha 1B-adrenoceptors expressed in transfected CHO cells and those natively expressed in DDT1 MF-2 hamster smooth muscle cells, and they provide additional information on the molecular mechanisms involved in agonist-induced regulation of alpha 1B-adrenoceptors.

6-Hydroxydopamine-mediated induction of rat brain metallothionein I mRNA.

Oxidative stress, resulting either from excess generation or reduced scavenging of free radicals, has been proposed to play a role in damaging striatal neurons in Parkinson's disease. Since metallothionein is able to regulate the intracellular redox potential, we have undertaken a group of experiments to learn whether or not 6-hydroxydopamine, which generates free radicals and is toxic to dopaminergic neurons, could alter the levels of zinc and metallothionein in the brain. The lesioning of the rat striatum with 6-hydroxydopamine (8.0 micrograms in 4 microliter 0.02% ascorbic acid) resulted in a reduction in the levels of zinc and metallothionein in the striatum but not other brain regions tested. However, the intracerebroventricular administration of 6-hydroxydopamine, in a dosage regimen that does not lesion catecholaminergic pathways but causes oxidative stress, enhanced dramatically the level of metallothionein I mRNA in some brain areas such as hippocampus, arcuate nucleus, choroid plexus, and granular layer of cerebellum, but not in the caudate putamen. The results of these studies are interpreted to suggest that zinc or metallothionein are altered in conditions where oxidative stress has taken place. Moreover, it is proposed that areas of brain, such as striatum containing high concentrations of iron, but low levels of inducible metallothionein are particularly vulnerable to oxidative stress.

Expression and regulation of brain metallothionein.

Many, but not all, zinc-containing neurons in the brain are a subclass of the glutamatergic neurons, and they are found predominantly in the telencephalon. These neurons store zinc in their presynaptic terminals and release it by a calcium-dependent mechanism. These "vesicular" pools of zinc are viewed as endogenous modulators of ligand- and voltage-gated ion channels. Metallothioneins (MTs) are low molecular weight zinc-binding proteins consisting of 25-30% cysteine, with no aromatic amino acids or disulfide bonds. The areas of the brain containing high contents of zinc such as the retina, the pineal gland, and the hippocampus synthesize unique isoforms of MT on a continuous basis. The four MT isoforms are thought to provide the neurons and glial elements with mechanisms to distribute, donate, and sequester zinc at presynaptic terminals; or buffer the excess zinc at synaptic junctions. In this cause, glutathione disulfide may participate in releasing zinc from MT. A similar nucleotide and amino acid sequence has made it difficult to obtain cDNA probes and antibodies capable of distinguishing indisputably among MT isoforms. MT-I and MT-II isoforms are found in the brain and in the peripheral tissues; MT-III isoform, possessing an additional seven amino acids, is expressed mostly in the brain and to a very minute extent in the intestine and pancreas; whereas MT-IV isoform is found in tissues containing stratified squamous epithelial cells. Since MTs are expressed in neurons that sequester zinc in their synaptic vesicles, the regulation of the expression of MT isoforms is extremely important in terms of maintaining the steady-state level of zinc and controlling redox potentials. The concentration of zinc has been shown to be altered in an extensive number of disorders of the central nervous system, including alcoholism. Alzheimer-type dementia, amyotrophic lateral sclerosis, Down's syndrome, epilepsy, Friedreich's ataxia, Guillaine-Barré syndrome, hepatic encephalopathy, multiple sclerosis, Parkinson's disease, Pick's disease, retinitis pigmentosa, retinal dystrophy, schizophrenia, and Wernicke-Korsakoff syndrome. The status of MT isoforms and other low molecular weight zinc-binding proteins in these conditions, diseases, disorders, or syndromes is being delineated at this time. Since several of these disorders, such as amyotrophic lateral sclerosis, are associated with oxidative stress, and since MT is able to prevent the formation of free radicals, it is believed that cytokine-induced induction of MT provides a long-lasting protection to avert oxidative damage.

Distribution of zinc metallothionein I mRNA in rat brain using in situ hybridization.

Metallothionein (MT) isoforms I and II were first identified and characterized in our laboratories in several regions of brain, in hippocampal neurons in primary culture, and in retinoblastoma and neuroblastoma cell lines. In this study, by having employed the MT-I cDNA as a probe, we sought to gain additional insight about the function of MT by discerning the regional distribution of its mRNA. Northern blot analyses of brain mRNA revealed that the administration of zinc enhanced dramatically MT-I mRNA (570 bp). The in situ hybridization study revealed that MT-I mRNA was located in several areas of brain, with the highest concentrations found in the cerebellum, hippocampus, and ventricles. The results of these studies are interpreted to suggest that zinc enhances the synthesis of MT mRNA and MT in turn may participate in zinc associated functions in neurons.

Cloning and expression of the alpha 2C-adrenergic receptor from the OK cell line.

The alpha 2-adrenergic receptors have been divided into four pharmacological subtypes, alpha 2A, alpha 2B, alpha 2C, and alpha 2D. The OK cell line, a cell line derived from an opossum kidney, expresses the alpha 2C-adrenergic receptor and is the prototypical cell line for the alpha 2C receptor subtype. The cloned human alpha 2C-C4 and rat RG10 receptors have been shown to express alpha 2C pharmacology. Here we report the cloning and expression of the OK alpha 2C-adrenergic receptor, OKc2. The receptor has 64% deduced amino acid identity and 21% similarity to the alpha 2-C4 receptor, giving an overall similarity of 85%. The clone, expressed in Chinese hamster ovary cells, has a pharmacology that correlates very well (r = 0.97) with that of the native OK cell alpha 2C-adrenergic receptor, and it is negatively coupled to adenylyl cyclase.

The cloning and expression of an OK cell cDNA encoding a 5-hydroxytryptamine1B receptor.

Serotonin (5-hydroxytryptamine or 5-HT) is an important biogenic amine that functions as both a neurotransmitter and a hormone in the central nervous system (CNS) and the periphery. We report here the isolation of a cDNA from the OK cell that encodes a serotonin receptor (OKc1). When expressed in cultured cells, it displayed the pharmacological profile and negative coupling with adenylyl cyclase characteristic of a 5-HT1B receptor subtype. Similar to the cloned rodent 5-HT1B receptors, it had high affinity for the beta-adrenergic ligand [125I]iodocyanopindolol, because of the presence of an asparagine instead of a threonine residue in the seventh transmembrane region. The ligands used displayed the following rank order of potencies: cyanopindolol > RU24969 > methiothepin > serotonin > sumatriptan > methysergide > 8-OH-DPAT > isoproterenol. This profile correlates well (r = 0.97) with the native OK cell 5-HT1B receptor. When OKc1 is compared to the rat, mouse, and human 5-HT1B receptors, it has an amino acid sequence identity of 82%, but it is only 54% identical to the human 5-HT1D receptor.

Pathogenesis of the B variant of encephalomyocarditis virus.

Variants of encephalomyocarditis virus (EMCV) are immunologically indistinguishable by hyperimmune serum, but, with the exception of EMCV-B, each produces a different disease syndrome and infects the central nervous system in mice infected via the intraperitoneal route of inoculation. The B variant is benign in that it does not produce any overt signs of infection at doses as high as 10(6) pfu per animal. The present study was carried out to determine if EMCV-B was pathogenic when administered via the intracranial route and, if so, to delineate the area(s) of the brain infected. The results show that, when given i.c., EMCV-B is similar to other variants of EMCV in that it infects and replicates in the brain, causing encephalitis, neuronal necrosis in Ammon's horn of the hippocampus, and clinical signs of infection. The data indicate that receptor sites for EMCV-B are present on brain cells and suggest that its benign nature when given by the intraperitoneal route reflects an inability to cross the blood-brain barrier.

Differences in the two-dimension-gel electrophoresis protein patterns of the lethal K and benign B variants of encephalomyocarditis virus.

Variants of encephalomyocarditis virus (EMCV) are indistinguishable by hyperimmune serum. In spite of their antigenic similarity, they produce different disease syndromes in susceptible strains of mice. To understand the basis for the diversity in pathogenicity, studies have been initiated to characterize each of the virus variants. In this study, two-dimensional gel electrophoresis was used to compare the proteins produced by the benign EMCV-B with those produced by lethal EMCV-K. The data show that (i) the replication cycle of each of the virus variants is characteristic of picornaviruses, (ii) the VP1 of EMCV-K is more basic than that of EMCV-B, and (iii) three proteins, one a major component of VP1, the other two with molecular weight of about 12,000, are present in EMCV-K but not in EMCV-B.

Cloning of minute virus of mice cDNAs and preliminary analysis of individual viral proteins expressed in murine cells.

cDNAs corresponding to RNA from the autonomous parvovirus minute virus of mice were cloned into constitutive and inducible expression vectors. These clones generate viral NS2, VP1, and VP2 proteins individually. Initial examination of these clones by transient expression analysis and analysis of stably transformed murine cell lines inducibly expressing these constructs indicated that they will be useful tools for characterizing the function of individual minute virus of mice gene products.

Tropism and histopathology of the D, B, K, and MM variants of encephalomyocarditis virus.

Variants of encephalomyocarditis virus (EMCV), which are immunologically indistinguishable by hyperimmune serum, produce different disease syndromes in mice. For instance, in ICR Swiss male mice, EMCV-B produces no overt illness, EMCV-MM produces severe neurological signs followed by death, EMCV-D destroys pancreatic beta cells producing a disease syndrome resembling insulin-dependent diabetes mellitus, and EMCV-K is lethal but produces no overt signs of infection. The present study was done to determine the tissue tropism and histopathology of each of these EMCV variants in the ICR Swiss mouse model. The data show the highest concentrations in the following organs: EMCV-D in the pancreas, EMCV-B in the pancreas, EMCV-MM in the cerebrum, and EMCV-K in the medulla/brainstem. They also show that the pathological lesions produced by each variant correlate well with viral titers.

Inhibition of virus-induced diabetes mellitus by interferon is influenced by the host strain.

The diabetogenic variant of encephalomyocarditis virus (EMC-D) induces a diabetes-like syndrome in certain strains of mice. A study was done to determine if virus-induced diabetes could be prevented by interferon (IFN). It was found that the production of diabetes by EMC-D was blocked by either IFN beta or a variety of IFN-inducers in SWR/J, but not ICR Swiss mice. The replication of EMC-D in cell culture was inhibited by IFN beta. It is concluded that the response of pancreatic beta cells to the protective effect of IFN, is probably under genetic control.