Outbreak of abdominal distension and obstipation in a C57BL/6J experimental autoimmune encephalomyelitis study.

Seventy-four 9-week old female C57BL/6J mice housed in a conventional facility were manipulated to induce experimental autoimmune encephalomyelitis, among which 26 developed clinical signs including lethargy, absence of defecation, and abdominal distension. By gross necropsy examination, there was distension of the cecum and colon with fecal impaction. By histologic examination, there was severe ulcerative and proliferative typhlocolitis. Fecal ELISA confirmed the presence of toxins A and B of Clostridium difficile. Alteration in immune status of the immunocompetent mice, due to stress caused by experimental manipulation or autoimmune disease, may have led to intestinal dysbiosis, followed by opportunistic infections resulting in C. difficile-associated disease. This report brings to light the occurrence of the disease in immunocompetent laboratory mice during experimental manipulations associated with alteration in immune status, and it discusses potential hazards associated with conventional housing within a hospital-associated research institute.

Eukaryotes versus prokaryotes: an estimate of evolutionary distance.

The divergence of nucleated organisms and bacteria was 2.6 times more remote in evolution than the divergences of the nucleated organisms into sparate kingdoms, as evidenced by genetic changes in cytochrome c and transfer RNA. The development of the genetic code through the differentiation of transfer RNA's for different amino acids was still more remote in evolution. The overall states of transfer RNA evolution in bacteria and nucleated organisms were comparable.

Naltrexone modulates tumor response in mice with neuroblastoma.

Naltrexone, an opiate antagonist, had both stimulatory and inhibitory effects, depending on the dosage, on the growth of S20Y neuroblastoma in A/Jax mice. Daily injections of 0.1 milligram of naltrexone per kilogram of body weight, which blocked morphine-induced analgesia for 4 to 6 hours per day, resulted in a 33 percent tumor incidence, a 98 percent delay in the time before tumor appearance, and a 36 percent increase in survival time. Neuroblastoma-inoculated mice receiving 10 milligrams of naltrexone per kilogram, which blocked morphine-induced analgesia for 24 hours per day, had a 100 percent tumor incidence, a 27 percent reduction in the time before tumor appearance, and a 19 percent decrease in survival time. Inoculation of neuroblastoma cells in control subjects resulted in 100 percent tumor incidence within 29 days. These results show that naltrexone can modulate tumor response and suggest a role for the endorphin-opiate receptor system in neuro-oncogenic events.

Increased brain size and cellular content in infant rats treated with an opiate antagonist.

From birth to day 21, rat offspring received daily injections of naltrexone at a dosage that blocked morphine-induced analgesia 24 hours a day. At 21 days, body, brain, and cerebellar weights of naltrexone-injected animals were 18, 11, and 5 percent greater than corresponding control weights. In addition, morphometric analysis of the cerebrum revealed a somatosensory cortex that was 18 percent thicker than that of the controls. The cerebellum of naltrexone-treated rats was 41 percent larger in total area and contained at least 70 percent more glial cells and 30 percent more granule neurons. Neurons derived prenatally were unaffected by drug treatment. These results show that naltrexone can stimulate body and brain growth in rats and suggest a role for the endorphin and opiate receptor system in development.

Distribution of enkephalin immunoreactivity in germinative cells of developing rat cerebellum.

The cellular distribution of enkephalin, an endogenous opioid, in the developing rat cerebellum was determined by immunocytochemistry. Methionine and leucine enkephalin were concentrated in the external germinal layer, a matrix of proliferative cells; staining was confined to the cortical cytoplasm. Enkephalin was not detected by immunocytochemistry in differentiated neural cells. These results indicate that endogenous opioids are involved specifically in early phases of nervous system development, particularly cell proliferation and differentiation.

Dimerisation of a chromo shadow domain and distinctions from the chromodomain as revealed by structural analysis.

BACKGROUND: Proteins such as HP1, found in fruit flies and mammals, and Swi6, its fission yeast homologue, carry a chromodomain (CD) and a chromo shadow domain (CSD). These proteins are required to form functional transcriptionally silent centromeric chromatin, and their mutation leads to chromosome segregation defects. CSDs have only been found in tandem in proteins containing the related CD. Most HP1-interacting proteins have been found to associate through the CSD and many of these ligands contain a conserved pentapeptide motif. RESULTS: The 1.9 A crystal structure of the Swi6 CSD is presented here. This reveals a novel dimeric structure that is distinct from the previously reported monomeric nuclear magnetic resonance (NMR) structure of the CD from the mouse modifier 1 protein (MoMOD1, also known as HP1beta or M31). A prominent pit with a non-polar base is generated at the dimer interface, and is commensurate with binding an extended pentapeptide motif. Sequence alignments based on this structure highlight differences between CDs and CSDs that are superimposed on a common structural core. The analyses also revealed a previously unrecognised circumferential hydrophobic sash around the surface of the CD structure. CONCLUSIONS: Dimerisation through the CSD of HP1-like proteins results in the simultaneous formation of a putative protein-protein interaction pit, providing a potential means of targeting CSD-containing proteins to particular chromatin sites.

Modulation of murine neuroblastoma in nude mice by opioid antagonists.

Naltrexone, an opioid antagonist, had an inhibitory effect on the growth of murine S20Y neuroblastoma in BALB/c nude mice. Daily injections of 0.1 mg naltrexone/kg, which invoked a receptor blockade for 6-8 hours/day, resulted in 31-92% delay in latency time prior to tumor expression and a 27-49% increase in mean survival time; the magnitude of antitumor response was governed by tumor burden. Inoculation of neuroblastoma (10(6)-2.5 X 10(4) cells) resulted in measurable tumors in 10-13 days and mean survival times of 30-34 days. Immunoreactive beta-endorphin was detected in tumor tissue (39.7 pg/mg protein). Receptor binding assays revealed specific saturable binding of ligands related to delta- and kappa-binding sites, but not for the mu-binding site. These results demonstrate that opioid antagonist modulation of neuro-oncogenesis is not dependent on the integrity of T-cell-mediated immunity and suggest the feasibility of utilizing the nude mouse model in exploring the role of endogenous opioids in human cancers.

Opioid receptors and endogenous opioids in diverse human and animal cancers.

Receptor binding studies demonstrated specific high-affinity, saturable binding of a number of opioid ligands to a wide variety of neural and nonneural human and animal tumors. Radioimmunoassays revealed the presence of beta-endorphin and methionine-enkephalin in these tumors. Both methionine- and leucine-enkephalin were detected in tumor tissue by immunocytochemistry, with immunoreactivity related to the cortical cytoplasm of tumor cells, but not to cell nuclei. Endogenous opioids and receptors were found in benign and malignant tumors representative of ectodermal, mesodermal, and endodermal origin. Receptors and endogenous opioid peptides were present in tumors from many different species, including those transplanted into nude mice. These results suggest that opioid receptors and endogenous opioids are fundamental features of human and animal cancers.

Specific labeling of O6-alkylguanine-DNA alkyltransferase by reaction with O6-(p-hydroxy[3H]methylbenzyl)guanine.

O6-Alkylguanine-DNA alkyltransferase (AGT) is an important DNA repair protein that removes adducts from the O6 position of guanine by transferring them to a cysteine residue within its amino acid sequence. Mammalian AGTs are readily inactivated by incubation with O6-benzyl-guanine (BG), which is an alternative substrate for the protein. To examine this inactivation in more detail and to develop a procedure for the specific labeling of human AGT, we synthesized a BG derivative, O6-(p-hydroxy[3H]methylbenzyl_guanine ([3H]HMBG) and examined its interaction with AGT in HT29 cell extracts and in HT29 cells. Incubation of human AGT with [3H]HMBG led to the incorporation of radioactivity in the protein in a time- and temperature-dependent manner. This reaction was specific, since neither AGT that was first inactivated by reaction with BG nor proteins other than AGT were labeled. Digestion of the labeled AGT with trypsin showed that a single peptide contained the label. Sequencing of this peptide indicated that the label was bound to cysteine-145. These results demonstrate that AGT accepts HMBG as a substrate and becomes inactivated by transfer of a p-hydroxymethylbenzyl residue to the cysteine-145 acceptor site. When [3H]HMBG was added to cultures of HT29 cells which are Mer+ and express active AGT, radioactivity was incorporated in a macromolecule and could be detected by autoradiography. No such labeling occurred with BE or CHO cells, which are Mer- and lack AGT. Examination of the interaction of [3H]HMBG with mutant AGT proteins that differ greatly in their abilities to react with BG showed that there was a strong correlation between the reaction with BG and the labeling with [3H]HMBG. These results indicate that [3H]HMBG is a potentially useful reagent for the detection and localization of AGT activity and for the investigation of its mechanism of action.

Crystallization of the complex of actin with gelsolin segment 1.

Crystals of a 1:1 complex between human gelsolin segment 1 and actin have been grown from solutions containing polyethylene glycol 6000. The crystals are orthorhombic, space group P2(1)2(1)2(1); the axes are a = 57.4 A, b = 70.4 A, c = 184.5 A. They are moderately stable to X-rays and diffract to beyond 2.5 A. There is one molecule of complex in the asymmetric unit.

Refined crystal structure of the phosphorylase-heptulose 2-phosphate-oligosaccharide-AMP complex.

The crystal structure of phosphorylase b-heptulose 2-phosphate complex with oligosaccharide and AMP bound has been refined by molecular dynamics and crystallographic least-squares with the program XPLOR. Shifts in atomic positions of up to 4 A from the native enzyme structure were correctly determined by the program without manual intervention. The final crystallographic R value for data between 8 and 2.86 A resolution is 0.201, and the overall root-mean-square difference between the native and complexed structure is 0.58 A for all protein atoms. The results confirm the previous observation that there is a direct hydrogen bond between the phosphate of heptulose 2-phosphate and the pyridoxal phosphate 5'-phosphate group. The close proximity of the two phosphates is stabilized by an arginine residue, Arg569, which shifts from a site buried in the protein to a position where it can make contact with the product phosphate. There is a mutual interchange in position between the arginine and an acidic group, Asp283. These movements represent the first stage of the allosteric response which converts the catalytic site from a low to a high-affinity binding site. Communication of these changes to other sites is prevented in the crystal by the lattice forces, which also form the subunit interface. The constellation of groups in the phosphorylase transition state analogue complex provides a structural basis for understanding the catalytic mechanism in which the cofactor pyridoxal phosphate 5'-phosphate group functions as a general acid to promote attack by the substrate phosphate on the glycosidic bond when the reaction proceeds in the direction of glycogen degradation. In the direction of glycogen synthesis, stereoelectronic effects contribute to the cleavage of the C-1-O-1 bond. In both reactions the substrate phosphate plays a key role in transition state stabilization. The details of the oligosaccharide, maltoheptaose, interactions with the enzyme at the glycogen storage site are also described.

Molecular and mutational analysis of a gelsolin-family member encoded by the flightless I gene of Drosophila melanogaster.

The flightless locus of Drosophila melanogaster has been analyzed at the genetic, molecular, ultrastructural and comparative crystallographic levels. The gene encodes a single transcript encoding a protein consisting of a leucine-rich amino terminal half and a carboxyterminal half with high sequence similarity to gelsolin. We determined the genomic sequence of the flightless landscape, the breakpoints of four chromosomal rearrangements, and the molecular lesions in two lethal and two viable alleles of the gene. The two alleles that lead to flight muscle abnormalities encode mutant proteins exhibiting amino acid replacements within the S1-like domain of their gelsolin-like region. Furthermore, the deduced intron-exon structure of the D. melanogaster gene has been compared with that of the Caenorhabditis elegans homologue. Furthermore, the sequence similarities of the flightless protein with gelsolin allow it to be evaluated in the context of the published crystallographic structure of the S1 domain of gelsolin. Amino acids considered essential for the structural integrity of the core are found to be highly conserved in the predicted flightless protein. Some of the residues considered essential for actin and calcium binding in gelsolin S1 and villin V1 are also well conserved. These data are discussed in light of the phenotypic characteristics of the mutants and the putative functions of the protein.

The cannabinoid CB1 antagonist AM 251 produces food avoidance and behaviors associated with nausea but does not impair feeding efficiency in rats.

RATIONALE: A growing body of evidence suggests that cannabinoid CB1 receptor antagonists have potential therapeutic utility as appetite suppressants. However, the specific mechanisms underlying the reduction in food intake produced by these drugs are not well understood. OBJECTIVE: Considering the known antiemetic and motor-suppressive effects of CB1 agonists, the present studies were conducted to determine if the reductions in food intake induced by the CB1 antagonist AM 251 could result from nausea or impairments in intake-related motor control, rather than solely from appetite suppression. METHODS: Three experiments were conducted to examine the effects of AM 251 (2.0, 4.0, or 8.0 mg/kg or vehicle) on detailed parameters of food intake, on the development of conditioned taste avoidance, and on taste reactivity. RESULTS: In the first experiment, acute administration of AM 251 dose-dependently decreased food intake; nevertheless, feeding rate (grams consumed per time spent eating) and food handling were unaffected, which suggests that food intake was not reduced because of severe motor impairments. In the second experiment, AM 251 dose-dependently reduced intake of a flavor with which it had previously been associated, indicating that conditioned taste avoidance had developed. Lastly, AM 251 was found to induce conditioned rejection reactions in a dose-dependent manner. CONCLUSIONS: The CB1 antagonist AM 251 may reduce food intake in part by inducing nausea or malaise, but not because of incoordination or motor slowing related to feeding.

The opioid growth factor, [Met5]-enkephalin, and the zeta opioid receptor are present in human and mouse skin and tonically act to inhibit DNA synthesis in the epidermis.

Opioid peptides serve as tonically active negative growth factors in neural and non-neural cells, in addition to being neuromodulators. To investigate the involvement of opioids in homeostatic renewal of epithelial cells in the epidermis, mice were given systemic injections of the potent opioid antagonist, naltrexone (NTX) (20 mg/kg). Disruption of opioid-receptor interaction by NTX resulted in an elevation of 42 and 72% in DNA synthesis in skin from the dorsum and plantar surface of the hindfoot, respectively, within 2 h; response to NTX was dependent on the circadian rhythm in each region examined. Injection of the naturally occurring and potent opioid growth factor (OGF), [Met5]-enkephalin, at 1 mg/kg depressed DNA synthesis in the dorsum and plantar surface by 42 and 19%, respectively, within 2 h; the effects of OGF complied with the pattern of circadian rhythm in each area of skin. The decreases in labeling index evoked by OGF were blocked by concomitant administration of the opioid antagonist, naloxone (10 mg/kg); naloxone alone at the dosage utilized had no influence on cell replicative processes. In tissue culture studies, OGF and NTX respectively depressed and elevated DNA synthesis. Both OGF and its receptor, zeta, were detected in all but the cornified layer of the epidermis in murine skin from the dorsum, plantar surface, pinnae, and tail. In addition, both peptide and receptor were observed in basal and suprabasal cells of the human epidermis. These results lead to the suggestion that an endogenous opioid peptide and its receptor are present and govern cellular renewal processes in the skin in a direct manner, regulating DNA synthesis in a tonically inhibitory, circadian rhythm-dependent fashion.

Opioids and the developing organism: a comprehensive bibliography, 1984-1988.

A comprehensive bibliography of the literature concerned with opioids and the developing organism for 1984-1988 is presented. Utilized with companion papers (Neurosci. Biobehav. Rev. 6:439-479; 1982; 8:387-403; 1984), these articles cover the clinical and laboratory references beginning in 1875. For the years 1984, 1985, 1986, 1987, and 1988, a total of 877 citations were recorded. A series of indexes accompanies the citations in order to make the literature more accessible. These indexes are divided into clinical and laboratory topics, and subdivided into such topics as the type of opioid explored and the general area of biological interest (e.g., physiology).

Opiates, endorphins, and the developing organism: a comprehensive bibliography, 1982-1983.

A comprehensive bibliography of the literature concerned with opiates, endorphins, and the developing organism for 1982 and 1983 is presented. Utilized with a companion paper (Neurosci Biobehav Rev 6: 439-479, 1982) these articles cover clinical and laboratory references beginning in 1875. For the years 1982 and 1983, a total of 385 citations was recorded. A series of indexes accompanies the citations in order to make the literature more accessible. These indexes are divided into clinical and laboratory topics. The clinical section is subdivided into: age of subject examined, maternal aspects, the fetus, and the offspring. The laboratory section is subdivided into: type of opiate/endorphin studied, species utilized, and major subject areas explored.

Opiates, endorphins and the developing organism: a comprehensive bibliography.

A comprehensive bibliography of the literature concerned with opiates, endorphins, and the developing organism is presented. A total of 1378 clinical and laboratory references, with citations beginning in 1875, are recorded. A series of indexed accompanies the citations in order to make the literature more accessible. These indexes are divided into clinical and laboratory topics. The clinical section is subdivided into: age of subject examined; maternal aspects; effects on the fetus; pharmacology, physiology, and the withdrawal syndrome; and "other" effects on the offspring. The laboratory section is subdivided into: type of opiate/endorphin studied; species utilized; and major subject areas explored.

The opioid growth factor, [Met5]-enkephalin, inhibits DNA synthesis during recornification of mouse tail skin.

Opioid peptides serve as tonically active negative growth regulators in renewing and regenerating epithelia. To examine the involvement of opioids in renewal of the stratum corneum after tape stripping of tail skin, C57BL/6 J mice were given systemic injections of the potent opioid antagonist, naltrexone (NTX, 20 mg/kg i.p.) following injury. Blockade of opioid-receptor interaction by NTX for 4 h resulted in an elevation of 36-66% in basal cell DNA synthesis measured 24 h after injury. Injection of the endogenous opioid peptide, [Met5]-enkephalin (OGF, 10 mg/kg i.p.) 4 h before termination, suppressed radiolabelled thymidine incorporation in the basal cell layer by 37-46% at 24 h after wounding. The magnitude of the effects on DNA synthesis of OGF, but not NTX, depended on the timing of administration with respect to injury. OGF maximally depressed basal cell labelling (72%) when given 16 h after tape stripping. Concomitant administration of naloxone (10 mg/kg) with OGF blocked the inhibition of DNA synthesis; naloxone alone at the dosage utilized had no effect on cell labelling. Both OGF and its receptor, OGFr, were detected by immunocytochemistry in the basal and suprabasal cell layers, but not the cornified layer of tape stripped and uninjured tail skin. These results indicate: (a) a native opioid peptide and its receptor are expressed in epidermal cells of injured and uninjured mouse tail skin; (b) removal of the stratum corneum by tape stripping does not disrupt the function of the endogenous opioid growth system; (c) the proliferative response to wounding of the tail is tonically inhibited by the receptor-mediated action of an endogenous opioid peptide; and (d) DNA synthesis by basal cells can be elevated by disrupting opioid peptide receptor interactions.

Temporal variation in cellular proliferation during recornification of mouse tail skin.

The influence of the time of injury on subsequent epidermal regeneration is unknown. Epidermal cell proliferation of tail skin in C57BL/6J mice in response to tape stripping was followed for 7 days by radiolabelled thymidine incorporation and autoradiography. The homeostatic labelling index (LI) of the basal epidermis of unmanipulated, unwounded (control) animals was 7.6% and did not vary depending on the time of day. Tape stripping increased the LI of epidermal basal cells 110% above control values 24 h after injury. Labelling indexes of epidermal basal cells in the skin adjacent to the wounded area were 7.0%. Basal cell DNA synthesis stimulated by wounding exhibited a distinct temporal variation at 24 h postinjury, with tail skin wounded at 12.00 h found to be 275% greater than control values and elevated 78% from LIs recorded at any other time point. This temporal spike was due to the time of day at which wounding occurred rather than the time point when the LI was determined. Mice wounded at 12.00 h and terminated 27 h later (15.00 h) had LIs that were 52% greater than wounds created at 09.00 h and examined at 12.00 h the following day. Higher levels of DNA synthesis in tail skin injured at 12.00 h compared to wounding at 09.00 h was detected 12-48 h after injury. Furthermore, DNA synthesis in wounds created at 12.00 h returned to baseline levels 1-2 days earlier than tail skin wounded at 09.00 h. Investigation of other strains of mice detected differences in radiolabelling of epidermal basal cells 24 h after tape stripping at 12.00 h or 09.00 h in CD-1 and BALB/cJ mice, but not in the C3H/HeJ strain. These results indicate: (a) there is no diurnal variation in the LI of mouse tail skin under normal homeostatic conditions (b) tape stripping is a potent stimulator of basal cell turnover in the epidermis (c) the time of wounding determines the magnitude of the increase in the LI of basal cells following injury, and (d) the proliferative response to wounding of the tail is dependent on the strain of mouse.