Effects of selective opioid agonists on feline colonic transit.

The mu agonist morphine and the non-specific opioid antagonist naloxone both may accelerate feline colonic transit; the effects of morphine are dose dependent. Kappa and delta receptor function was studied in the present work. Colonic transit of a radionuclide marker instilled into the cecum was quantitated for 6 hr in a crossover study. The delta agonist [D-Pen2,D-pen5]enkephalin (1 mg/kg, i.m.) prolonged the cecum and ascending colon half-emptying time by 337% (P less than 0.05), and delayed the progression of the geometric center over time. The kappa agonist U-50,488 (1 mg/kg, i.m.) had no apparent effect on the cecum and ascending colon, but delayed filling of the descending colon. Loperamide, an antidiarrheal agent, also delayed colonic transit. Thus, selective opioid agonists have both site and functional differences in their effect on feline colonic transit.     

Cytochemical localization of plasma membrane ATPase activity in plant cells

Summary The cytochemical localization of ATPase activity has been investigated in maize root cells using both lead and cerium-based capture methods. With both methods, staining at the plasma membrane was observed in all cells of the root, although the precipitate obtained with cerium was more uniform and granular than that with lead. Controls using no substrate or no magnesium, -glycerophosphate to replace ATP, vanadate or boiled tissue generally showed little or no staining. However, biochemical studies on purified plasma membrane fractions showed that ATPase activity was markedly inhibited by fixation, particularly by glutaraldehyde, and also by lead and cerium ions. Non-enzymic hydrolysis of ATP by cerium was greater than that by lead. The value and limitations of these procedures for the localization of plasma membrane H⁺-ATPase activity are summarized in relation to previous criticisms of these methods.Abbreviations DTT dithiothreitol - EDTA ethylene diaminetetraacetic acid - GP B-glycerophosphate - PCMBS p-chloromercuribenzene sulphonic acid - PMSF phenylmethylsulphonyl fluoride     

Insulin, glucagon and somatostatin localization in the pancreas of metamorphosed Xenopus laevis

The current study was designed to determine if insulin, glucagon and somatostatin-containing cells are present in the pancreas of adult Xenopus laevis. Localization methods utilized included cytochemical aldehyde fuchsin (AF) staining as well as the immunochemical peroxidase antiperoxidase (PAP) procedure for light microscopy. The results show numerous large clusters of AF-positive cells within a network of highly vascularized acinar tissue. PAP immunochemical localization with insulin antibody on adjacent sections demonstrates positive immunoreactivity to AF-positive cell groups and also the presence of immunoreactive insulin (IRI). Cells exhibiting this immunoreactivity are located in the central region of the islet-like structures. Serial sections not only show PAP immunoreactivity for IRI, but also for immunoreactive glucagon (IRG) and immunoreactive somatostatin (IRS) in the same islet-like structure. IRG and IRS-containing cells are situated around the periphery of the islet-like structures, surrounding the central core of IRI-containing cells. Antibody specificity was confirmed by homologous and heterologous antigen immuno-absorbance assays, as well as incubation of adjacent sections in preimmune sera. Based on this data we conclude that: the distribution of cells of the endocrine pancreas of metamorphosed Xenopus laevis is similar to that of many mammals and certain urodeles. Given the apparent specificity of the antigen-antibody reactions, it appears that Xenopus insulin, glucagon and somatostatin are structurally conserved.     

Physicochemical and structural studies onAcinetobacter calcoaceticus RAG-1 and MR-481—Two standard strains in hydrophobicity tests

Acinetobacter calcoaceticus RAG-1 and MR-481, two standard strains used in microbial adhesion to hydrocarbons (MATH), were characterized by contact angles, pH-dependent zeta potentials, elemental surface composition by X-ray photoelectron spectroscopy (XPS), and molecular composition by infrared spectroscopy (IR). Negatively stained (methylamine tungstate) and ruthenium red-stained cells were studied by transmission electron microscopy to reveal the absence or presence of surface appendages. Despite the fact thatA. calcoaceticus RAG-1 is known to be extremely hydrophobic in MATH, whereas MR-481 is a completely non-hydrophobic mutant, neither XPS nor IR indicated a significant difference in chemical composition of the cell surfaces. Contact angles with polar liquids, water and formamide, were considerably higher on RAG-1 than on MR-481, in accordance with their relative hydrophobicities as measured by MATH. However, no significant differences in contact angles were observed between the two strains with apolar liquids like diiodomethane,-bromonaphthalene, and hexadecane. Fibrous extensions on RAG-1, observed after ruthenium red staining, were absent on the non-hydrophobic mutant MR-481. Tentatively, these extensions could be held responsible for the hydrophobicity ofA. calcoaceticus RAG-1.     

The involvement of ethanol in the free radical reaction of 6-hydroxydopamine

ESR spin trapping technique was used to detect and analyze free radical formation. When 6-hydroxydomine (6-OHDA) was incubated alone or in the presence of a free radical generating system (H₂O₂ and FeSO₄), hydroxyl free radicals were observed in a concentration-dependent manner. Glutathione was found to be the most effective scavenger of the ESR signal when compared with vitamin E or Mannitol. The addition of ethanol resulted in the formation of the pure hydroxyethyl free radicals. The amount of hydroxyethyl free radicals in the system was dependent upon the concentration of ethanol and the formation of hydroxyethyl free radicals correlated well with the extent of lipid peroxidation and the loss of enzymic activity of the membrane-bound (Na⁺, K⁺)-ATPase. We suggest that in the biological system ethanol may potentiate the neurotoxicity of 6-OHDA with the formation of hydroxyethyl free radicals, which are longer-lived and far more damaging to membranes that the hydroxyl radicals. These data lead us to further hypothesize that the neuronal degeneration caused by 6-OHDA and other compounds that generate free radicals could be potentiated in the presence of ethanol.     

Chromatid repulsion associated with Roberts/SC phocomelia syndrome is reduced in malignant cells and not expressed in interspecies somatic-cell hybrids.

Different cell types from a female patient with Roberts/SC phocomelia syndrome were evaluated quantitatively for the presence of repulsion of heterochromatin and satellite regions of mitotic chromosomes. Whereas EBV-transformed lymphoblasts from an established cell line revealed these phenomena at frequencies equal to those in PHA-stimulated lymphocytes and cultured skin fibroblasts, aneuploid cells from a metastatic melanoma displayed them at 50% lower frequency. Cocultivation of the patient's fibroblasts with either an immortal Chinese hamster cell line or with a human male fibroblast strain carrying a t(4;6)(p14;q21) translocation showed that the phenomenon was not corrected or induced by a diffusible factor or by cell-to-cell contact. In each experiment, only the patient's metaphase spreads revealed chromatid repulsion. In fusion hybrids between the patient's fibroblasts and an established Chinese hamster cell line, the human chromosomes behaved perfectly normally, suggesting that the gene product which is missing or mutant in Roberts/SC phocomelia syndrome is supplied by the Chinese hamster genome.     

Tubulin pseudogenes as markers for hominoid divergence

Processed pseudogenes arise via unimolecular events that result in the integration of nonfunctional (and therefore non-selected) regions of DNA into the germ line. The sequence of such pseudogenes can be used as a novel form of evolutionary clock: the older a particular pseudogene, the more mutations it has acquired relative to the selectively constrained functional gene from which it was originally derived. We have used specific beta-tubulin gene probes to assay for the presence of fully sequenced processed pseudogenes in genomic DNA from various hominoid species. The data suggest that orangutan is more closely related to human, chimpanzee and gorilla than is generally believed.     

Molecular cloning and DNA sequence analysis of genes encoding cytotoxic T lymphocyte-defined HLA-A3 subtypes: the E1 subtype

Influenza-specific cytotoxic T cells restricted by HLA-A3 and allogeneic CTL specific for HLA-A3 recognize differences between serologically indistinguishable HLA-A3 antigens. Previous biochemical studies have indicated that such differential recognition can be explained by alterations in the primary structure of class I heavy chains. Characterization of these sequence differences may therefore identify portions of the class I molecule that form determinants recognized by CTL. In this study, we describe the cloning and sequencing of an HLA-A3 subtype from donor E1 (E1-A3). Cloning of the gene encoding E1-A3 was simplified by determining that a 15.5-kb BamHI fragment contains the complete gene and is characteristic of HLA-A3 and only one other class I gene (HLA-A11). Comparison of the E1-A3 sequence to that of a previously sequenced HLA-A3 gene for exons encoding extracellular class I domains revealed three nucleotide differences. All of these differences were located within a discrete region of exon 3 (encoding the alpha 2 domain) and result in a change of two amino acids, at positions 152 (Glu----Val) and 156 (Leu----Gln). This finding suggests that these amino acids are crucial for the information of a determinant recognized by CTL. Furthermore, the altered nucleotide sequence of E1-A3 is identical to the sequence of the HLA-Aw24 gene for codons 128 to 161. These observations of multiple clustered changes in the E1-A3 subtype (relative to the prototype sequence) and identity of the altered sequence with the sequence of another class I gene support the concept that gene conversion is a primary mechanism for the generation of class I polymorphism.     

Crystallization and preliminary crystallographic data of the Fab fragment of an anti-phenylalanine hydroxylase monoclonal antibody

Two crystal habits, one rod shaped and the other square prismatic, of the Fab fragment of a monoclonal anti-phenylalanine hydroxylase antibody have been grown using the method of vapour phase diffusion against polyethylene glycol 6000. The square prisms diffract to better than 2.8 A, belong to the space group P1 and have unit cell parameters a = 41.8 A, b = 50.3 A, c = 114.7 A, alpha = 97.6 degrees, beta = 91.7 degrees, gamma = 91.0 degrees, while the rod-shaped crystals belong to the space group P212121, have unit cell parameters a = 105.6 A, b = 119.8 A, c = 82.2 A and diffract to 3.5 A resolution.     

Secretory bursts of growth hormone secretion in the dog may be initiated by somatostatin withdrawal

In 28 6-h experiments on 10 conscious resting trained male dogs, plasma growth hormone (GH) was determined at 5-min intervals by radioimmunoassay. For all experiments, the basal GH concentration in plasma was 0.80 +/- 0.06 ng mL-1. In each experiment, 1-3 secretory bursts of GH occurred, raising plasma GH 2.4 to 15.3 times basal concentrations (for all 43 bursts, 6.6 +/- 0.4 times the basal value). Metabolic clearance rates (MCR) and apparent distribution volumes (V) were determined, using stepwise infusions of canine GH. The MCR (3.99 +/- 0.30 mL kg-1 min-1) and V (57.9 +/- 5.5 mL kg-1) were used to transform the GH concentration versus time data into GH secretion rates, using a single compartment approach. Basal GH secretion rates for all 28 experiments were 3.12 +/- 0.24 ng kg-1 min-1. The secretory bursts yield peak GH secretion rates of 9.4 +/- 0.8 times basal secretion and these steep-sloped bursts last 25.1 +/- 1.2 min. Six-hour infusions of 0.15 microgram kg-1 min-1 of somatostatin (SRIF) abolished all secretory bursts but did not lower basal secretion rates. In five of seven SRIF infusion experiments in which samples were taken after the infusion ceased a secretory burst was seen in the hour following cessation of infusion (in four cases within 10 min). These secretory bursts lasted 23.0 +/- 2.9 min and were similar to those seen in control experiments. Infusions of SRIF at 0.05 microgram kg-1 min-1 had no effect. These results imply that during basal GH secretion, a surfeit of SRIF impinges on the somatotrophs, as extra SRIF does not further lower basal secretion. However, during secretory bursts, very little SRIF must be present, as exogenous SRIF blocks these bursts. The bursts are similar in duration to overshoots provoked in perifused dispersed rat somatotrophs by removal of an SRIF signal. It seems likely that their cause in vivo is similar. (All values are means +/- SEM.)