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Biomedical subjects

A Zimmer

Publications and source records attributed to A Zimmer.

At least 55 records · Page 3Linked to original sources
PubMed1999

Increased mortality, hypoactivity, and hypoalgesia in cannabinoid CB1 receptor knockout mice.

Delta9-Tetrahydrocannabinol (Delta9-THC), the major psychoactive ingredient in preparations of Cannabis sativa (marijuana, hashish), elicits central nervous system (CNS) responses, including cognitive alterations and euphoria. These responses account for the abuse potential of cannabis, while other effects such as analgesia suggest potential medicinal applications. To study the role of the major known target of cannabinoids in the CNS, the CB1 cannabinoid receptor, we have produced a mouse strain with a disrupted CB1 gene. CB1 knockout mice appeared healthy and fertile, but they had a significantly increased mortality rate. They also displayed reduced locomotor activity, increased ring catalepsy, and hypoalgesia in hotplate and formalin tests. Delta9-THC-induced ring-catalepsy, hypomobility, and hypothermia were completely absent in CB1 mutant mice. In contrast, we still found Delta9-THC-induced analgesia in the tail-flick test and other behavioral (licking of the abdomen) and physiological (diarrhea) responses after Delta9-THC administration. Thus, most, but not all, CNS effects of Delta9-THC are mediated by the CB1 receptor.

Animals↗
PubMed1999

Altered gene expression in striatal projection neurons in CB1 cannabinoid receptor knockout mice.

The basal ganglia, a brain structure critical for sensorimotor and motivational aspects of behavior, contain very high levels of CB1 cannabinoid receptors. These receptors are activated by endogenous lipophilic ligands, and they are thought to mediate behavioral effects of cannabinoid drugs. To evaluate the role of the endogenous cannabinoid system in the regulation of basal ganglia pathways, we have investigated the effects of targeted deletion of CB1 receptors on gene expression of various neuropeptides and transmitter-related enzymes in basal ganglia neurons. Mice without CB1 receptors are extremely hypoactive in a test for exploratory behavior (open-field test), showing markedly reduced locomotion and rearing. These CB1 mutants display significantly increased levels of substance P, dynorphin, enkephalin, and GAD 67 mRNAs in neurons of the two output pathways of the striatum that project to the substantia nigra and the globus pallidus. Our findings demonstrate that elimination of CB1 receptors results in behavioral abnormalities and functional reorganization of the basal ganglia.

Animals↗
PubMed1999

Region-specific up-regulation of opioid receptor binding in enkephalin knockout mice.

Mu and delta opioid receptors were labeled in enkephalin knockout mice by quantitative autoradiography. Discrete, large increases (100-300%) were found in limbic forebrain structures for mu binding and striatum and pallidum for delta binding. The up-regulation of opioid receptors may reflect a form of 'denervation supersensitivity. ' The receptor up-regulation in limbic areas is consistent with the increased emotional and aggressive behaviors observed in the enkephalin knockout mice.

Animals↗
PubMed1999

Antisense oligonucleotide delivery with polyhexylcyanoacrylate nanoparticles as carriers.

Polyalkylcyanoacrylate nanoparticles are effective colloidal drug carriers and were prepared by an emulsion polymerization process. Antisense oligonucleotides were loaded on the particles by adsorption. A cationic polymer, DEAE-dextran, was incorporated into the particle matrix or a cationic hydrophobic detergent (CTAB) was used to form a lipophilic oligonucleotide ion pair. Enzymatic digestion of the oligonucleotides was almost quantitatively inhibited by this nanoparticle complex and cellular uptake by different cell lines was significantly enhanced. In vivo the biodistribution of the oligonucleotide nanoparticle complex resulted in targeting of oligonucleotides to the liver. Improvements in antisense treatments with nanoparticles were demonstrated for tumor therapy as well as for antiviral applications.

Animals↗
PubMed1999

The patched signaling pathway in tumorigenesis and development: lessons from animal models.

The identification of mutations in the human homolog of the Drosophila segment polarity gene Patched in basal cell carcinoma has sparked intense interest in the role of this gene in human disorders. The transmembrane protein Patched is a receptor for the morphogene Sonic Hedgehog. Sonic Hedgehog/Patched signaling involves another transmembrane protein, Smoothened, and its intracellular effectors, including the proto-oncogene GLI1. During the past 2 years it has become evident that mutations in Patched or in one of the components of its signaling pathway contribute to the formation of several common human tumors. It is now well established that Patched is a tumor suppressor gene. The Sonic Hedgehog/Patched/Smoothened signaling pathway is thus rapidly emerging as one of the most important regulators of oncogenic transformation. This pathway also plays an important role during mammalian embryonic development. This dual role is especially visible in humans with inherited Patched mutations. Such patients suffer from Gorlin, or nevoid basal cell carcinoma, syndrome and exhibit a variety of developmental defects accompanied by a predisposition to tumor formation. Activating mutations in Sonic Hedgehog and Smoothened lead to similar phenotypes as do loss-of function mutations in Patched. By means of transgenic and gene targeting technologies the respective mutations have been expressed in the mouse. Such mutant mouse strains exhibit many symptoms observed in humans. These strains are useful models to study the pathogenesis of several common human tumors and developmental defects. Furthermore they provide important tools to study the Sonic Hedgehog/Patched/Smoothened signaling at the molecular and biochemical level.

Animals↗
PubMed1999

Synthesis of cholesterol modified cationic lipids for liposomal drug delivery of antisense oligonucleotides.

The paper describes a novel synthesis of cholest-5-en-3 beta-yl-6-aminohexyl ether (AH-Chol). AH-Chol was used to prepare positively charged liposomes. The liposomes consisted of phospholipon 90H and the cationic cholesterol derivative in an equimolar ratio. Liposome preparation was achieved by membrane homogenization after rehydration of a dry lipid film. Oligonucleotides (ODN) were adsorbed to the cationic liposomes very efficiently. At an ODN/liposome ratio of 1:5 (10:50 micrograms/ml) 84.2 +/- 5.4% of the ODNs were bound to the liposomal membrane. Within the range of 1:40 and 1:100 charge neutralization occurred and the liposome dispersion showed an increase in particle size due to aggregation. Below or above this range of charge neutralization the ODN loaded liposome preparation was physically stable, no sedimentation, increase of vesicle size or vesicle aggregation occurred.

Cholesterol↗
PubMed1999

Evaluation of aminoalkylmethacrylate nanoparticles as colloidal drug carrier systems. Part I: Synthesis of monomers, dependence of the physical properties on the polymerization methods.

Conventional nanoparticles based on acrylic compounds are lipophilic and possess a negative surface charge. This is due to their manufacturing process and to the chemical structure of the polymer. Hence, these particles are not suitable for the adsorption of hydrophilic anionic drugs. In the present investigation, positively charged copolymer nanoparticles prepared from aminoalkyl- and methylmethacrylates were evaluated, with regard to their physical properties. This report provides a detailed description of the synthesis of the non-commercially available monomers and their polymerization procedure. Various parameters were investigated, such as comonomer content, total amount of monomer, concentration of the radical initiator, and the composition of the polymerization medium. The resulting particle diameter and the surface charge were found to be strongly dependent on the polymerization conditions and on the pH. Optimization of the polymerization procedure yielded nanoparticles of about 200 nm exhibiting a positive surface charge. The charges of the different copolymer particles were then compared at different pH values. N-trimethylaminoethylmethacrylate (TMAEMC) nanoparticles with quaternary ammonium groups located at their surfaces, possessed a nearly constant positive zeta potential at various pH values and, consequently, pH-independent particle diameters. The physical characteristics of the other aminoalkyl copolymers correlated with the basicity of the monomers employed and were found to be strongly dependent on the pH of the dispersion medium. Aminoethylmethacrylate (AEMC), methylaminoethylmethacrylate (MMAEMC), and aminohexylmethacrylate (AHMC) as well as aminoethylmethacrylamide (AHMAC) copolymer nanoparticles exhibited a strong positively charged surface even at physiological pH and, therefore, are useful candidates for the adsorption of anionic drugs.

Biocompatible Materials↗
PubMed1999

Evaluation of aminoalkylmethacrylate nanoparticles as colloidal drug carrier systems. Part II: characterization of antisense oligonucleotides loaded copolymer nanoparticles.

Aminoalkylmethacrylate methylmethacrylate copolymer nanoparticles were evaluated for their use as potential drug carrier systems. Their cytotoxicity, as well as the loading of antisense oligonucleotides that were employed as anionic model drugs depended on the substitution of the basic aminoalkyl copolymer. Toxic influences on the integrity of cell membranes depended on aminoalkyl groups located on the particle surfaces. Toxicity was observed either by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assays using African green monkey kidney (AGMK) cells or by a hemolysis test, where the efflux of haemoglobin from disrupted erythrocytes was measured. The cytotoxic effects were increased by the elongation of the N-alkyl chain by four additional methylene groups. Lipophilic polymethylmethacrylate (PMMA) homopolymer nanoparticles showed a negative surface charge and, therefore, were not suitable for the adsorption of anionic drugs. The surface charge was changed to positive values by the incorporation of basic monomers. Consequently, the loading efficacy was increased by raising the basic copolymer portion. Additionally, a pH-dependent loading behaviour of oligonucleotides was observed. Substitution of the amino nitrogen protons by methyl groups led to a decreased oligonucleotide loading and to a reduced cytotoxicity. Nanoparticles with permanent positively charged quarternary ammonium groups showed a high pH-independent loading efficacy, but also possessed a high cytotoxic potential. In this study, cationic copolymer nanoparticles containing 30% (w/w) methylaminoethyl-methacrylate (MMAEMC) were found to be optimal with regard to biocompatibility and carrier properties for hydrophilic anionic antisense oligonucleotides. A significant portion of adsorbed oligonucleotides were protected from enzymatic degradation. The cellular uptake of oligonucleotides into Vero cells was significantly enhanced by this methylaminoethyl-methacrylate derivative.

Animals↗
PubMed1999

Preproenkephalin is a Th2 cytokine but is not required for Th2 differentiation in vitro.

Preproenkephalin (PPNK) mRNA expression has been detected in many cells of the immune system, including monocytes and lymphocytes. In the present paper, the expression of PPNK mRNA in purified CD4+ Th1 and Th2 lymphocyte subpopulations is investigated and correlated with the presence of the opioid neuropeptides leu- and met-enkephalin. We found that PPNK mRNA and met-enkephalin were present at higher levels in the Th2 cultures compared with the Th1 cultures. Lymphocytes from PPNK-deficient mice were then used to look at the role of PPNK in Th2 lymphocyte differentiation. Lymphocytes from these mice could be driven into a Th2 phenotype, suggesting that cultures containing IL-4 do not require PPNK for Th2 differentiation.

Animals↗
PubMed1999

Dose-related opposite modulation by nociceptin/orphanin FQ of substance P nociception in the nociceptors and spinal cord.

We previously reported that the intraplantar (i.pl.) application of nociceptin/orphanin FQ (N/OFQ) at extremely low doses elicited a nociception through a substance P (SP) release from nociceptor endings. In the present study, the nociception induced by SP (and N/OFQ) was abolished by intrathecal (i.t.) injection of neurokinin(1) (SP receptor) antagonist, suggesting the involvement of the stimulation of nociceptive primary SP neuron and SP release into spinal synapses. On the other hand, similar low doses of N/OFQ (i.t.) exerted nociceptive responses, characterized by scratching, biting, and licking, and these responses were blocked by an neurokinin(1) antagonist (i.t.) or capsaicin pretreatment or in tachykinin 1 gene knockout mice (tac1(-/-) mice), suggesting that N/OFQ receptor (NOR) also exists on the spinal terminals of SP neurons. When wide ranges of N/OFQ doses were used, a typical bell-shaped dose-response relationship was observed in both peripheral and central nociception tests. Furthermore, N/OFQ (1 nmol) administered i.pl. blocked SP (i.pl.)-induced flexor responses, which were abolished by pertussis toxin pretreatment or in NOR gene knockout (NOR(-/-)) mice. On the other hand, N/OFQ administered i.t. blocked SP (i.t.)-induced scratching, biting, and licking in capsaicin-pretreated and tac1(-/-) mice, and this antinociception was abolished in NOR(-/-) mice. All these findings suggest that N/OFQ has biphasic actions depending on doses in the nociceptors and spinal synapses and has postsynaptic antinociceptive actions in spinal cord by modulating SP signaling.

Analgesics↗
PubMed1999

Oligonucleotide and plasmid DNA packaging into polyoma VP1 virus-like particles expressed in Escherichia coli.

The drug delivery system described here is based on the properties of the capsoid or capsid-like structure resulting from the assembly of polyoma virus capsid protein VP1 expressed in Escherichia coli. The capsid protein VP1 was expressed as a fusion protein with a completely removable N-terminal His6 affinity tag. The pentameric morphology of the recombinant VP1 protein was confirmed by electron microscopy after affinity chromatography and factor Xa cleavage under conditions of low ionic strength. The self-assembly of VP1 capsoids can be induced from purified VP1 pentamers by increasing the ionic strength with (NH4)2SO4. These VP1 capsoid particles were packed in vitro with anti-sense oligonucleotides and plasmid DNA. The loading with DNA was pH-dependent. We observed the highest efficiency at pH 5. DNase I treatment of particles with encapsidated material showed that 37-55% of the bound oligonucleotides and fragments of 1.5-1.8 kb double-stranded DNA were protected against degradation.

Base Sequence↗
PubMed1998

Nociceptin/orphanin FQ-induced nociceptive responses through substance P release from peripheral nerve endings in mice.

We have studied the in vivo signaling mechanisms involved in nociceptin/orphanin FQ (Noci)-induced pain responses by using a flexor-reflex paradigm. Noci was 10,000 times more potent than substance P (SP) in eliciting flexor responses after intraplantar injection into the hind limb of mice, but the action of Noci seems to be mediated by SP. Mice pretreated with an NK1 tachykinin receptor antagonist or capsaicin, or mice with a targeted disruption of the tachykinin 1 gene no longer respond to Noci. The action of Noci appears to be mediated by the Noci receptor, a pertussis toxin-sensitive G protein-coupled receptor that stimulates inositol trisphosphate receptor and Ca2+ influx. These findings suggest that Noci indirectly stimulates nerve endings of nociceptive primary afferent neurons through a local SP release.

Animals↗
PubMed1998

Hypoalgesia in mice with a targeted deletion of the tachykinin 1 gene.

The tachykinin neuropeptides, substance P and substance K, are produced in nociceptive primary sensory neurons and in many brain regions involved in pain signaling. However, the precise role and importance of these neuropeptides in pain responses has been debated. We now show that mice that cannot produce these peptides display no significant pain responses following formalin injection and have an increased pain threshold in the hotplate test. On the other hand, the mutant mice react normally in the tail flick assay and acetic acid-induced writhing tests. These results demonstrate that substance P and/or substance K have essential functions in specific responses to pain.

Acetic Acid↗
PubMed1998

Targeted reduction of oxytocin expression provides insights into its physiological roles.

Oxytocin is a nonapeptide hormone that participates in the regulation of parturition and lactation. It has also been implicated in various behaviors, such as mating and maternal, and memory. To investigate whether or not oxytocin (OT) is essential for any of these functions, we eliminated, by homologous recombination, most of the first intron and the last two exons of the OT gene in mice. Those exons encode the neurophysin portion of the oxytocin preprohormone which is hypothesized to help in the packaging and transport of OT. The homozygous mutant mice have no detectable neurophysin or processed oxytocin in the paraventricular nucleus, supraoptic nucleus or posterior pituitary. Interestingly, homozygous mutant males and females are fertile and the homozygous mutant females are able to deliver their litters. However, the pups do not successfully suckle and die within 24 hours without milk in their stomachs. OT injection into the dams or rescue with the rat OT gene restores the milk ejection in response to suckling. OT is also needed for post-partum alveolar proliferation. These results indicate an absolute requirement for oxytocin for successful milk ejection, but not for mating, parturition and milk production, in mice. Furthermore, homozygous mutant mice show reduced aggression in some tests.

Aggression↗
PubMed1998

Craf-1 protein kinase is essential for mouse development.

The three mammalian Raf serine/threonine protein kinases mediate the transduction of proliferative and differentiative signals from a variety of cell surface receptors to the nucleus. We report here that Craf-1 is essential for mouse development, as its mutation results in embryonic lethality. Developmental defects are found in mutant placentas as well as in the skin and in the lungs of mutant embryos. Craf-1 mutants also display a generalized growth retardation which is consistent with the ubiquitous expression of Craf-1 and which could be due to the reduced proliferation of mutant cells. Interestingly, the time-point of embryonal death varies depending on the genetic background. This suggests that Craf-1-mediated signaling is affected by genetic background-specific alleles of other genes.

Abnormalities, Multiple↗
PubMed1998

Rhabdomyosarcomas and radiation hypersensitivity in a mouse model of Gorlin syndrome.

Gorlin (or nevoid basal cell carcinoma) syndrome is characterized by a variety of clinical problems including generalized overgrowth of the body, cysts, developmental abnormalities of the skeleton and a predisposition to benign and malignant tumors. The syndrome results from germline mutations of the human homolog of the drosophila segment polarity gene patched (ptc). Here we report that mice heterozygous for ptc develop many of the features characteristic of Gorlin syndrome and that they exhibit a high incidence of rhabdomyosarcomas (RMS), the most common soft-tissue sarcoma in children. The downstream signalling partner of ptc, gli1, was overexpressed in all RMSs analyzed, indicating that abnormal signalling of the ptc-gli1 pathway may be common for the various tumors associated with the syndrome. igf2, implicated in the formation of RMSs, was also overexpressed, suggesting cross-talk between the ptc and igf2 pathways in tumorigenesis. Developmental defects in Gorlin syndrome resemble those induced by ionizing radiation. We show that ptc heterozygous mice exhibit increased incidence of radiation-induced teratogenesis. This suggests a role for ptc in the response to ionizing radiation and provides a model for both the systemic (developmental) and stochastic (cancer) abnormalities observed in Gorlin syndrome.

Animals↗