Showing papers in "Journal of Biomedical Science in 2000"

Actions of melatonin in the reduction of oxidative stress

Abstract: Melatonin was discovered to be a direct free radical scavenger less than 10 years ago. Besides its ability to directly neutralize a number of free radicals and reactive oxygen and nitrogen species, it stimulates several antioxidative enzymes which increase its efficiency as an antioxidant. In terms of direct free radical scavenging, melatonin interacts with the highly toxic hydroxyl radical with a rate constant equivalent to that of other highly efficient hydroxyl radical scavengers. Additionally, melatonin reportedly neutralizes hydrogen peroxide, singlet oxygen, peroxynitrite anion, nitric oxide and hypochlorous acid. The following antioxidative enzymes are also stimulated by melatonin: superoxide dismutase, glutathione peroxidase and glutathione reductase. Melatonin has been widely used as a protective agent against a wide variety of processes and agents that damage tissues via free radical mechanisms.

Mitochondrial role in life and death of the cell.

Abstract: Mitochondria are the major ATP producer of the mammalian cell. Moreover, mitochondria are also the main intracellular source and target of reactive oxygen species (ROS) that are continually generated as by-products of aerobic metabolism in human cells. A low level of ROS generated from the respiratory chain was recently proposed to take part in the signaling from mitochondria to the nucleus. Several structural characteristics of mitochondria and the mitochondrial genome enable them to sense and respond to extracellular and intracellular signals or stresses in order to sustain the life of the cell. It has been established that mitochondrial respiratory function declines with age, and that defects in the respiratory chain increase the production of ROS and free radicals in mitochondria. Within a certain concentration range, ROS may induce stress responses of the cell by altering the expression of a number of genes in order to uphold energy metabolism to rescue the cell. However, beyond this threshold, ROS may elicit apoptosis by induction of mitochondrial membrane permeability transition and release of cytochrome c. Intensive research in the past few years has established that mitochondria play a pivotal role in the early phase of apoptosis in mammalian cells. In this article, the role of mitochondria in the determination of life and death of the cell is reviewed on the basis of recent findings gathered from this and other laboratories.

Neutralizing antibody provided protection against enterovirus type 71 lethal challenge in neonatal mice.

Abstract: Experimental infection with enterovirus type 71 (EV71) induced death in neonatal mice in an age- and dose-dependent manner. The mortality rate was 100% following intraperitoneal inoculation 1-day-old ICR mice and this gradually decreased as the age at the time of inoculation increased (60% in 3-day-old mice and no deaths occurred in mice older than 6 days of age). A lethal dose greater than 108 PFU was necessary. Lethargy, failure to gain weight, rear limb tremors and paralysis were observed in the infected mice before death. EV71 was isolated from various tissues of the dead mice. Using a reverse transcription polymerase chain reaction technique with a specific primer pair, a 332-bp product was detected in the tissues that produced a culture positive for EV71. Protection against EV71 challenge in neonatal mice was demonstrated following passive transfer of serum from actively immunized adult mice 1 day after inoculation with the virus. Pups from hyperimmune dams were resistant to EV71 challenge. Additionally, maternal immunization with a formalin-inactivated whole-virus vaccine prolonged the survival of pups after EV71 lethal challenge.

Nitric oxide and the cerebral vascular function.

Abstract: The presence of a cholinergic vasodilator innervation to cerebral circulation is well established. Despite its high endogenous concentration in cerebral blood vessels, acetylcholine (ACh) is not the transmitter for vasodilation. This finding has led to the discovery that nitric oxide (NO), which is coreleased with ACh and neural peptides such as vasoactive intestinal polypeptide (VIP) from the respective cholinergic-nitrergic (nitric oxidergic) nerves and the VIPergic-nitrergic nerves, is the primary transmitter in relaxing smooth muscle. ACh and VIP act presynaptically to inhibit and facilitate, respectively, the release of NO. Release of NO from cerebral vascular endothelial cells is also well established. A similar system for recyclingL-citrulline toL-arginine for synthesizing more NO has been demonstrated in both cerebral perivascular nerves and endothelial cells. Neuronal and endothelial NO appears to play an important role in controlling cerebral vascular tone and circulation in health and disease.

Adeno-Associated Virus-Based Vectors in Gene Therapy

Abstract: Adeno-associated virus (AAV) vectors were shown capable of high efficiency transduction of both dividing and nondividing cells and tissues. AAV-mediated transduction leads to stable, long-term transgene expression in the absence of apparent immune response. These properties and the broad host range of AAV vectors indicate that they constitute a powerful tool for gene therapy purposes. An additional potential benefit of AAV vectors is their ability to integrate site-specifically in the presence of Rep proteins which can be expressed transiently, thus limiting their suspected adverse effects. The major restrictions of AAV as vectors are their limited genetic capacity and strict packaging size constraint of less than 5 kb. Another difficulty is the labor-intensive and expensive procedure for the production and packaging of recombinant AAV vectors. The major benefits and drawbacks of AAV vectors and advances made in the past 3 years are discussed.

Age-Associated Changes of Superoxide Dismutase and Catalase Activities in the Rat Brain

Abstract: Oxygen free radicals have been proposed to be involved in the process of aging. Superoxide dismutase (SOD) and catalase are important for antioxidative defense. In this study, profiles of SOD, catalas

p53 gene status modulates the chemosensitivity of non-small cell lung cancer cells

Abstract: This study examined the effects of p53 gene status on DNA damage-induced cell death and chemosensitivity to various chemotherapeutic agents in non-small cell lung cancer (NSCLC) cells. A mutant p53 gene was introduced into cells carrying the wild-type p53 gene and also vice versa to introduce the wild-type p53 gene into cells carrying the mutant p53 gene. Chemosensitivity and DNA damage-induced apoptosis in these cells were then examined. This study included five cell lines, NCI-H1437, NCI-H727, NCI-H441 and NCI-H1299 which carry a mutant p53 gene and NCI-H460 which carries a wild-type p53 gene. Mutant p53-carrying cells were transfected with the wild-type p53 gene, while mutant p53 genes were introduced into NCI-H460 cells. These p53 genes were individually mutated at amino acid residues 143, 175, 248 and 273. The representative cell line NCI-H1437 cells transfected with wild-type p53 gene (H1437/wtp53) showed a dramatic increase in susceptibility to three anticancer agents (7-fold to cisplatin, 21-fold to etoposide, and 20-fold to camptothecin) compared to untransfected or neotransfected H1437 cells. An increase in chemosensitivity was also observed in wild-type p53 transfectants of H727, H441, H1299 cells. The results of chemosensitivity were consistent with the observations on apoptotic cell death. H1437/wtp53 cells, but not H1437 parental cells, exhibited a characteristic feature of apoptotic cell death that generated oligonucleosomal-sized DNA fragments. In contrast, loss of chemosensitivity and lack of p53-mediated DNA degradation in response to anticancer agents were observed in H460 cells transfected with mutant p53. These observations suggest that the increase in chemosensitivity was attributable to wild-type p53 mediation of the process of apoptosis. In addition, our results also suggest that p53 gene status modulates the extent of chemosensitivity and the induction of apoptosis by different anticancer agents in NSCLC cells.

Itch-scratch responses induced by opioids through central mu opioid receptors in mice.

Abstract: We examined scratch-inducing effects of intracisternal, intrathecal and intradermal injections of morphine and some opioid agonists in mice. Intracisternal injection of morphine (3 nmol/animal) and the μ-receptor agonist [D-Ala2, N-Me-Phe4, Gly5-ol]enkephalin (DAMGO; 0.2 nmol/animal) elicited scratching of the face, with little effect on scratching of the trunk. Intracisternal injection of the δ-receptor agonist [D-Pen2,5]enkephalin (DPDPE) and the κ-receptor agonist U50488 were without effects. Intrathecal injection of morphine (0.1–3 nmol/animal) produced a dose-dependent increase in body scratching, with little effects on face scratching. Face scratching induced by intrathecal morphine (3 nmol/animal) was almost abolished by subcutaneous pretreatment with naloxone (1 mg/kg). Intradermal injections of morphine (3–100 nmol/site), DAMGO (1–100 nmol/site), DPDPE (10 and 100 nmol/site) and U50488 (10–100 nmol/site) did not elicit scratching of the site of injection. Intradermal injection of histamine (100 nmol/site) induced the scratching in ICR, but not ddY, mice and serotonin (30 and 50 nmol/site) elicited the scratching in either strain of mice. The results suggest that opioids induce scratching, and probably itching, through central μ-opioid receptors in the mouse.

Delta Opioid Peptide[D- Ala2,D-Leu5]Enkephalin Promotes Cell Survival

Abstract: By studying the hibernation in ground squirrels, a protein factor termed hibernation induction trigger (HIT) was found to induce hibernation in summer-active ground squirrels. Further purification of HIT yielded an 88-kD peptide that is enriched in winter hibernator. Partial sequence of the 88-kD protein indicates that it may be related to the inhibitor of metalloproteinase. Delta opioid [D-Ala2,D-Leu5]enkephalin (DADLE) also induced hibernation. HIT and DADLE were found to prolong survival of peripheral organs preserved en bloc or as a single preparation. These organs include the lung, the heart, liver and kidney. DADLE also promotes survival of neurons in the central nervous system. Methamphetamine (METH) is known to cause destruction of dopaminergic (DA) terminals in the brain. DADLE blocked and reversed the DA terminal damage induced by METH. DADLE acted against this effect of METH at least in part by attenuating the mRNA expressions of a tumor necrosis factor p53 and an immediate early gene c-fos. DADLE also blocked the neuronal damage induced by ischemia-reperfusion following a transient middle cerebral artery occlusion. In PC12 cells, DADLE blocked the cell death caused by serum deprivation in a naltrexone-sensitive manner. Thus, DADLE, and by extension the endogenous delta opioid peptides and delta opioid receptors, may play an important role in organ and neuronal survival. Here, critical developments concerning these fascinating cell protective properties of DADLE are reviewed.

Preventive and therapeutic vaccines for human papillomavirus-associated cervical cancers.

Abstract: ‘High risk’ genotypes of the human papillomavirus (HPV), particularly HPV type 16, are the primary etiologic agent of cervical cancer. Thus, HPV-associated cervical malignancies might be prevented or treated by induction of the appropriate virus-specific immune responses in patients. Sexual transmission of HPV may be prevented by the generation of neutralizing antibodies that are specific for the virus capsid. In ongoing clinical trials, HPV virus-like particles (VLPs) show great promise as prophylactic HPV vaccines. Since the capsid proteins are not expressed at detectable levels by basal keratinocytes, therapeutic vaccines generally target other nonstructural viral antigens. Two HPV oncogenic proteins, E6 and E7, are important in the induction and maintenance of cellular transformation and are coexpressed in the majority of HPV-containing carcinomas. Therefore, therapeutic vaccines targeting these proteins may provide an opportunity to control HPV-associated malignancies. Various candidate therapeutic HPV vaccines are currently being tested whereby E6 and/or E7 are administered in live vectors, in peptides or protein, in nucleic acid form, as components of chimeric VLPs, or in cell-based vaccines. Encouraging results from experimental vaccination systems in animal models have led to several prophylactic and therapeutic vaccine clinical trials. Should they fulfill their promise, these vaccines may prevent HPV infection or control its potentially life-threatening consequences in humans.

Age-Associated Changes in Interferon-γ and Interleukin-4 Secretion by Purified Human CD4+ and CD8+ T Cells

Abstract: Aging is associated with a decline in immune function. Interferon-γ (IFN-γ) and interleukin-4 (IL-4), two important immune deviation-related cytokines, are mainly produced by type 1 and type 2 T cells, respectively. To investigate the age-associated changes in the secretion of these two cytokines, 20 elderly and 20 young subjects fulfilling the SENIEUR protocol were enrolled. The ratios of CD4+ to CD8+ T cells were not different between the two age groups. The CD4+ and CD8+ T cells were purified by a magnetic cell sorting system, and then activated by concurrent anti-CD3 and anti-CD28 stimulation. The released cytokines were determined by ELISA. Both the CD4+ and the CD8+ T cells of the elderly individuals secreted a significantly larger amount of IFN-γ after activation. Profound IL-4 production by CD8+ T cells was observed in the older subjects compared with that of the young subjects. These data suggested that age-associated decrease in immunity may be related to an imbalance in the secretion of immune deviation cytokines. The number of IL-4-secreting CD8+ T cells (T cytotoxic 2) rose significantly in the older individuals. Our design also provided a useful way to differentiate the T cell subsets secreting the same cytokine, such as IFN-γ-producing T helper 1 and T cytotoxic 1 cells.

Genetic reassortment and patch repair by recombination in retroviruses.

Abstract: Retroviral particles contain a diploid RNA genome which serves as template for the synthesis of double-stranded DNA in a complex process guided by virus-encoded reverse transcriptase. The dimeric nature of the genome allows the proceeding polymerase to switch templates during copying of the copackaged RNA molecules, leading to the generation of recombinant proviruses that harbor genetic information derived from both parental RNAs. Template switching abilities of reverse transcriptase facilitate the development of mosaic retroviruses with altered functional properties and thereby contribute to the restoration and evolution of retroviruses facing altering selective forces of their environment. This review focuses on the genetic patchwork of retroviruses and how mixing of sequence patches by recombination may lead to repair in terms of re-established replication and facilitate increased viral fitness, enhanced pathogenic potential, and altered virus tropisms. Endogenous retroelements represent an affluent source of functional viral sequences which may hitchhike with virions and serve as sequence donors in patch repair. We describe here the involvement of endogenous viruses in genetic reassortment and patch repair and review important examples derived from cell culture and animal studies. Moreover, we discuss how the patch repair phenomenon may challenge both safe usage of retrovirus-based gene vehicles in human gene therapy and the use of animal organs as xenografts in humans. Finally, the ongoing mixing of distinct human immunodeficiency virus strains and its implications for antiviral treatment is discussed.

Inhibition of ras oncogene: a novel approach to antineoplastic therapy.

Abstract: The most frequently detected oncogene alterations, both in animal and human cancers, are the mutations in theras oncogene family. These oncogenes are mutated or overexpressed in many human tumors, with a high incidence in tumors of the pancreas, thyroid, colon, lung and certain types of leukemia. Ras is a small guanine nucleotide binding protein that transduces biological information from the cell surface to cytoplasmic components within cells. The signal is transduced to the cell nucleus through second messengers, and it ultimately induces cell division. Oncogenic forms of p21ras lead to unregulated, sustained signaling through downstream effectors. Theras family of oncogenes is involved in the development of both primary tumors and metastases making it a good therapeutic target. Several therapeutic approaches to cancer have been developed pointing to reducing the altered gene product or to eliminating its biological function: (1) gene therapy with ribozymes, which are able to break down specific RNA sequences, or with antisense oligonucleotides, (2) immunotherapy through passive or active immunization protocols, and (3) inhibition of p21ras farnesylation either by inhibition of farnesyl transferase or synthesis inhibition of farnesyl moieties.

Investigations of the effects of ethanol on warfarin binding to human serum albumin.

Abstract: Ethanol effects on warfarin binding to human serum albumin (HSA) have been studied by equilibrium dialysis and fluorescence methods at pH 7.4 in phosphate-buffered saline at 37°C. In the presence of v

Transcription Factors Involved in Pancreatic Islet Development

Abstract: The endocrine pancreas is an organ of enormous importance, since its dysfunction causes diabetes, one of the most common human diseases in the world Regulation of pancreatic endocrine cell determination and differentiation requires a unique set of transcription factors, including basic helix-loop-helix and homeodomain-containing proteins The physiological role of individual transcription factor has been characterized by gene disruption in the mouse The results indicate that these genes are not only involved in tissue-specific activation of downstream target genes for islet-specific hormones, but also critical for the proper islet morphogenesis Future elucidation of the genetic relationship of these genes will lead to a better understanding of the molecular mechanisms controlling endocrine pancreas formation and will contribute to the development of new therapeutic approaches to diabetes

Role of flhDC in the Expression of the Nuclease Gene nucA, Cell Division and Flagellar Synthesis in Serratia marcescens

Abstract: We investigated in Serratia marcescens the functions of the flhDC operon, which controls motility and cell division in enteric bacteria. Included in our evaluatio

Supraspinal circuitry mediating opioid antinociception: antagonist and synergy studies in multiple sites.

Abstract: Supraspinal opioid antinociception is mediated by sensitive brain sites capable of supporting this response following microinjection of opioid agonists. These sites include the ventrolateral periaqueductal gray (vIPAG), the rostral ventromedial medulla (RVM), the locus coeruleus and the amygdala. Each of these sites comprise an interconnected anatomical and physiologically relevant system mediating antinociceptive responses through regional interactions. Such interactions have been identified using two pharmacological approaches: (1) the ability of selective antagonists delivered to one site to block antinociception elicited by opioid agonists in a second site, and (2) the presence of synergistic antinociceptive interactions following simultaneous administration of subthreshold doses of opioid agonists into pairs of sites. Thus, the RVM has essential serotonergic, opioid, cholinergic and NMDA synapses that are necessary for the full expression of morphine antinociception elicited from the vIPAG, and the vIPAG has essential opioid synapses that are necessary for the full expression of opioid antinociception elicited from the amygdala. Further, the vIPAG, RVM, locus coeruleus and amygdala interact with each other in synergistically supporting opioid antinociception.

DNA vaccination using the fragment C of botulinum neurotoxin type A provided protective immunity in mice

Abstract: Botulinum neurotoxin (BoNT) is one of the most toxic substances known to produce severe neuromuscular paralysis. The currently used vaccine is prepared mainly from biohazardous toxins. Thus, we studied an alternative method and demonstrated that DNA immunization provided sufficient protection against botulism in a murine model. A plasmid of pBoNT/A-Hc, which encodes the fragment C gene of type A botulinum neurotoxin, was constructed and fused with an Igκ leader sequence under the control of a human cytomegalovirus promoter. After 10 cycles of DNA inoculation with this plasmid, mice survived lethal doses of type A botulinum neurotoxin challenges. Immunized mice also elicited cross-protection to the challenges of type E botulinum neurotoxin. This is the first study demonstrating the potential use of DNA vaccination for botulinum neurotoxins.

Mutation L210W of HIV-1 reverse transcriptase in patients receiving combination therapy. Incidence, association with other mutations, and effects on the structure of mutated reverse transcriptase.

Abstract: Mutation L210W of HIV-1 reverse transcriptase (RT) is one of the six main mutations that confer in vivo resistance to zidovudine. Surprisingly, this mutation has received scant appraisal and its contribution to the genotypic resistance to nucleoside analogs is not well understood. The aim of this study was: (1) to study the frequency of mutation L210W in a large collection of HIV-1 sequences (2,049 samples, including 395 DNA and 1,654 RNA sequences) from patients receiving combination therapy, and (2) to analyze its association with the other mutations that confer resistance to zidovudine. A mutation at codon 210 (mainly L210W) was found in 647 (32%) of the 2,049 sequences analyzed. Only 43 (<7%) of these 647 genomes were also mutated at codon 70 (p < 10−5). In contrast, 98% of these 647 sequences were also mutated at codon 215 (essentially T215Y/F), and 94% at codon 41 (mainly M41L). These data showing a close association between L210W, T215Y/F, and M41L, and a mutual exclusion between K70R and L210W, were confirmed by analyzing the sequences stored in the HIV-1 sequences available through the Stanford HIV RT and Protease Database. Follow-up studies demonstrated that L210W appeared always after T215Y/F. This observation is consistent with crystallographic studies which suggested that the aromatic side chain of Trp 210 could stabilize the interaction of Phe/Tyr215 with the dNTP-binding pocket. This molecular cross-talk between amino acid chains occurs nearby the conserved Asp113 residue. Since the lateral chain of Arg70 may also interact with Asp113, this is likely to create a sterical hindrance around this residue. Thus, the R→K reversion of codon 70 may represent a compensatory mechanism allowing a functional rearrangement of the dNTP-binding pocket in the mutated RT.

Spinal plasticity of acute opioid tolerance

Abstract: Spinal acute opioid tolerance remains mechanistically undercharacterized. Expanded clinical use of direct spinal administration of opioids and other analgesics indicates that studies to further understand spinal mechanisms of analgesic tolerance are warranted. Rodent models of spinal administration facilitate this objective. Specifically, acute spinal opioid tolerance in mice presents a plasticity-dependent, rapid, and efficient opportunity for evaluation of novel clinical agents. Similarities between the pharmacology of acute and chronic spinal opioid tolerance, neuropathic pain, and learning and memory suggest that this model may serve as a high through-put predictor of bioactivity of novel plasticity-modifying compounds.

Molecular Characterization of a Hepatitis E Virus Isolate from Namibia

Abstract: Hepatitis E virus (HEV) causes sporadic and epidemic acute viral hepatitis in many developing countries. In Africa, hepatitis E has been documented by virus detection (reverse transcriptase polymerase chain reaction, RT-PCR) in Egypt, Chad, Algeria, Morocco and Tunisia. Cases of presumptive hepatitis E also have been documented by detection of antibody to HEV in the Sudan, Kenya, Ethiopia, Somalia, Djibouti and South Africa. Recently, we reported the recovery of 9 isolates of HEV from feces collected during an outbreak of jaundice in Namibia. These specimens were stored frozen for many years at the South African Institute for Medical Research awaiting new methods to determine the etiology of jaundice. HEV genomic sequences were detected by antigencapture RT-PCR with primers that amplified 2 independent regions of the HEV genome (ORF-2 and ORF-3). To further characterize the HEV 83-Namibia isolates, we determined the nucleotide (nt) sequence of the 3′ end of the capsid gene (296 of 1,980 nt in ORF-2) and ORF-3 for 1 isolate. The capsid gene sequence shared 86% identity with the prototype Burma strain and up to 96% identity with other African strains at the (nt) level, and 99% identity with Burma or other Africa strains at the amino acid level. A 188 (nt) fragment amplified from ORF-3 was also highly homologous to other HEV but was too short for meaningful comparison. Phylogenetic analysis indicated that HEV 83-Namibia is closely related to other African isolates, and differs from Burmese, Mexican and Chinese HEV. These data link the HEV causing the 1983 Namibia outbreak to more recent HEV transmission in northern and sub-Saharan Africa, suggesting this subgenotype of HEV is firmly established throughout the continent.

Characterization of TRBP1 and TRBP2. Stable stem-loop structure at the 5' end of TRBP2 mRNA resembles HIV-1 TAR and is not found in its processed pseudogene.

Abstract: TRBP1 and TRBP2 cDNAs have been isolated based on the ability of the protein that they encode to bind HIV-1 TAR RNA. The two cDNAs have different 5' end-termini resulting in 21 additional amino acids for TRBP2 protein compared to TRBP1. The corresponding gene is conserved in mammalian species. By PCR amplification of a human library, we have isolated an additional 22 nucleotides in the 5' end of TRBP2 cDNA. Based on the addition of these 22 new nucleotides, the first 87 nucleotides of TRBP2 mRNA can fold into a stable stem-loop structure that resembles TAR RNA. We have also isolated the DNA sequence that represents the TRBP processed pseudogene. The absence of full alignment between TRBP2 full-length cDNA and this sequence suggests that the stem-loop structure could have prevented a complete reverse transcription during pseudogene formation. Using different antibodies, three forms of TRBP can be identified in primate cells at 40, 43 and 50 kD, suggesting a differential expression from the cDNAs and post-translational modifications. Both TRBP1 and TRBP2 activate the basal and the Tat-activated level of the HIV-1 LTR in human and murine cells. Our data indicate that TRBP proteins act at a level prior to Tat function. TRBP could contribute to improved HIV expression in murine models.

Novel Signaling Pathways Contributing to Vascular Changes in Hypertension

Abstract: In hypertension, increased peripheral resistance maintains elevated levels of arterial blood pressure. The increase in peripheral ressitance results, in part, from abnormal constrictor and dilator responses and vascular remodeling. In this review, we consider four cellular signaling pathways as possible explanations for these abnormal vascular responses: (1) augmented signaling via the epidermal growth factor receptor to cause remodeling of the cerebrovasculature; (2) reduced sphingolipid signaling leading to blunted vasodilation and increased smooth muscle proliferation; (3) increased signaling via Rho/Rho kinase leading to enhanced vasoconstriction, and (4) a relative state of microtubular depolymerization favoring vasoconstriction in hypertension. These novel cell signaling pathways provide new pharmacological targets to reduce total peripheral vascular resistance in hypertension.

G protein activation by endomorphins in the mouse periaqueductal gray matter.

Abstract: The midbrain periaqueductal gray matter (PAG) is an important brain region for the coordination of μ-opioid-induced pharmacological actions. The present study was designed to determine whether newly isolated μ-opioid peptide endomorphins can activate G proteins through μ-opioid receptors in the PAG by monitoring the binding to membranes of the non-hydrolyzable analog of GTP, guanosine-5′-O-(3-[35S]thio)triphosphate ([35S]GTPγS). An autoradiographic [35S]GTPγS binding study showed that both endomorphin-1 and -2 produced similar anatomical distributions of activated G proteins in the mouse midbrain region. In the mouse PAG, endomorphin-1 and -2 at concentrations from 0.001 to 10 μM increased [35S]GTPγS binding in a concentration-dependent manner and reached a maximal stimulation of 74.6 ± 3.8 and 72.3 ± 4.0%, respectively, at 10 μM. In contrast, the synthetic selective μ-opioid receptor agonist [D-Ala2,NHPhe4,Gly-ol]enkephalin (DAMGO) had a much greater efficacy and produced a 112.6 ± 5.1% increase of the maximal stimulation. The receptor specificity of endomorphin-stimulated [35S]GTPγS binding was verified by coincubating membranes with endomorphins in the presence of specific μ-, δ- or κ-opioid receptor antagonists. Coincubation with selective μ-opioid receptor antagonists β-funaltrexamine or D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Phe-Thr-NH2 (CTOP) blocked both endomorphin-1 and-2-stimulated [35S]GTPγS binding. In contrast, neither δ- nor κ-opioid receptor antagonist had any effect on the [35S]GTPγS binding stimulated by either endomorphin-1 or -2. These findings indicate that both endomorphin-1 and -2 increase [35S]GTPγS binding by selectively stimulating μ-opioid receptors with intrinsic activity less than that of DAMGO and suggest that these new endogenous ligands might be partial agonists for μ-opioid receptors in the mouse PAG.

Modulatory effects of the human heat shock protein 70 on DNA vaccination.

Abstract: DNA vaccination with the plasmid expressing Japanese encephalitis virus (JEV) nonstructural protein 1 (pJNS1) has been shown to induce effective immunity against JEV infection. To further increase the efficacy of pJNS1 DNA vaccination, we coinjected pJNS1 with a plasmid that expresses heat shock protein 70.1 (pHSP70.1) into mice. We found that coinjection of pHSP70.1 enhanced both T cell proliferation and cytotoxic effects, but not the antibody response to JEV. Moreover, mice immunized with both pHSP70.1 and pJNS1 were resistant to lethal challenges of JEV, indicating that the protective immunity against JEV is not decreased, in spite of the low antibody titer via the immunization of pHSP70.1. Since DNA vaccination administered by pJNS1 did not elicit strong cellular immunity in our previous study, the administration of pHSP70.1 apparently could be used as an adjuvant to enhance cell-mediated immunity in this model system. Thus, coadministration of pHSP70.1 DNA with plasmid DNA encoding tumor- or virus-specific antigens might be very useful in the treatment of cancers and other infectious diseases.

Effect of prenatal opioid exposure on cholinergic development

Abstract: Opioid drugs such as methadone or buprenorphine are often used in the management of pregnant addicts. These drugs are generally thought of as nonteratogenic and preferable to repeated cycles of withdrawal in utero. However, evidence exists that perinatal exposure to these opioids delays and disrupts cholinergic development, particularly in the striatum. Acetylcholine (ACh) content and the expression of choline acetyltransferase protein and mRNA are reduced in the early postnatal period by prenatal opioid exposure in the rat. Although these indicators of the cholinergic phenotype return to normal levels over time, the activity of the cholinergic neurons remains disrupted, with a large increase in ACh turnover rate. The mechanism of these effects is unknown, but may involve changes in the expression of nerve growth factor, which is reduced by opioid exposure.

Protein oxidation and turnover.

Abstract: All biomacromolecules are faced with oxidative stress Oxidation of a protein molecule always induces inactivation of the molecule and introduces a tag to that molecule These modified protein molecules are prone to degradation in vivo by the proteasome system Coupling of protein modification and degradation of chemically modified proteins is one of the normal protein turnover pathways in vivo We call this a ‘chemical apoptosis’ process, which is one of the early manifestations of programmed cell death Impairment of the proteasome system leads to accumulation of modified nonfunctional proteins or ‘aged proteins’ that might cause various clinical syndromes including cataractogenesis, premature aging, neurological degeneration and rheumatoid disease The metal-catalyzed oxidation of biomacromolecules provides an excellent artificial aging system in vitro The system is very useful in the characterization of structure and function relationships of proteins (enzymes), especially in those containing metal binding domain(s), because the oxidation is always followed by an affinity cleavage at the metal binding site(s) that allows easy identification and further characterization

Parabrachial nucleus induces suppression of baroreflex bradycardia by the release of glutamate in the rostral ventrolateral medulla of the rat.

Abstract: The involvement of glutamatergic neurotransmission in the rostral ventrolateral medulla (RVLM) in the suppression of baroreflex bradycardia by the parabrachial nucleus (PBN) was investigated. Repeated electrical activation of the PBN increased the concentration of glutamate in the dialysate collected from the RVLM. The same stimulation also suppressed baroreflex bradycardia in response to transient hypertension evoked by phenylephrine (5 µg/kg, intravenously). Microinfusion ofL-glutamate (10, 50 or 100 µM) via the microdialysis probe into the RVLM dose-dependently elicited a significant inhibition of baroreflex bradycardia that paralleled the concentration and time course of the PBN-elicited elevation in extracellular glutamate in the RVLM. The suppression of baroreflex bradycardia elicited by microinjection ofL-glutamate (1 nmol) into the RVLM was appreciably reversed by coinjection of the NMDA receptor antagonist, dizocilpine (500 pmol), or the non-NMDA receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (50 pmol). These results suggest that an increase in the extracellular concentration of glutamate and activation of both NMDA and non-NMDA receptors in the RVLM may mediate the suppression of baroreflex bradycardia by activation of the PBN.

Antagonism of nitrous oxide antinociception in mice by intrathecally administered antisera to endogenous opioid peptides.

Abstract: Previously it was demonstrated that nitrous oxide antinociception in the mouse abdominal constriction test is mediated by κ-opioid receptors. Since nitrous oxide is thought to cause the neuronal release of endogenous opioid peptide to stimulate opioid receptors, this study was designed to identify the opioid peptides involved, especially in the spinal cord, by determining whether nitrous oxide antinociception can be differentially inhibited by intrathecally (i.t.) administered antisera to different opioid peptides. Male NIH Swiss mice were pretreated i.t. with rabbit antisera to opioid peptides then exposed 24 h later to one of three different concentrations of nitrous oxide in oxygen. Dose-response curves constructed from the data indicated that the antinociceptive effect of nitrous oxide was significantly antagonized by antisera to various dynorphins (DYNs) and methionine-enkephalin (ME), but not by antiserum to β-endorphin (β-EP). The AD50 values for nitrous oxide antinociception were significantly elevated by antisera to DYNs and ME but not β-EP. These findings of this study support the hypothesis that nitrous oxide antinociception in the mouse abdominal constriction test involves the neuronal release of DYN and ME in the spinal cord.

Endomorphins: Localization, Release and Action on Rat Dorsal Horn Neurons

Abstract: Endomorphin (Endo) 1 and 2, two tetrapeptides isolated from the bovine and human brain, have been proposed to be the endogenous ligand for the μ-opiate receptor. A multi-disciplinary study was undertaken to address the issues of localization, release and biological action of Endo with respect to the rat dorsal horn. First, immunohistochemical studies showed that Endo-1- or Endo-2-like immunoreactivity (Endo-1- or Endo-2-LI) is selectively expressed in fiber-like elements occupying the superficial layers of the rat dorsal horn, which also exhibit a high level of μ-opiate receptor immunoreactivity. Second, release of immunoreactive Endo-2-like substances (irEndo) from the in vitro rat spinal cords upon electrical stimulation of dorsal root afferent fibers was detected by the immobilized antibody microprobe technique. The site of release corresponded to laminae I and II where the highest density of Endo-2-LI fibers was localized. Lastly, whole-cell patch clamp recordings from substantia gelatinosa (SG) neurons of rat lumbar spinal cord slices revealed two distinct actions of exogenous Endo-1 and Endo-2: (1) depression of excitatory and/or inhibitory postsynaptic potentials evoked by stimulation of dorsal root entry zone, and (2) hyperpolarization of SG neurons. These two effects were prevented by the selective μ-opiate receptor antagonist β-funaltrexamine. The localization of endomorphin-positive fibers in superficial layers of the dorsal horn and the release of irEndo upon stimulation of dorsal root afferents together with the observation that Endo inhibits the activity of SG neurons by interacting with μ-opiate receptors provide additional support of a role of Endo as the endogenous ligand for the μ-opiate receptor in the rat dorsal horn.