Inhibitory effects of mersalyl and of antibodies directed against smooth-muscle myosin on a calcium adenosine triphosphatase of the plasma membrane from mouse liver cells.

The effect of mersalyl and of antibodies, directed against smooth-muscle myosin and skeletal muscle myosin, on the (Ca²⁺ + Mg²⁺)-activated adenosine triphosphatase (Ca,Mg)ATPase) system of mouse liver plasma membranes has been studied. Antismooth-muscle myosin inhibited by 38.6% at optimum substrate concentration the (Ca,Mg)ATPase with a K_m of 0.88 × 10^?3m. Mersalyl (0.5 mm) also inhibited this enzyme, the percentage inhibition being 44.6% at optimal substrate concentration. These results suggest the presence of a smooth-muscle myosin-like protein in the plasma membrane of mouse liver cells which has an associated (Ca,Mg)ATPase activity.     

Tissue fixation and osmium black formation with nonvolatile octavalent osmium compounds

Summary Several compounds of osmium^VIII, including potassium osmiamate and coordination complexes of OsO₄ with ammonia and various heterocyclic nitrogen compounds, have been synthesized and characterized. They have also been evaluated as substitutes for OsO₄ in postfixation of biological specimens and in light and electron microscopic cytochemical methods resulting in osmium black formation.The most useful of these osmic compounds, a molecular addition complex of hexamethylenetetramine (methenamine) with OsO₄, has a negligible vapor pressure of OsO₄. It has the molecular formula C₆H₁₂N₄.2OsO₄ and has been designated osmeth. Although it has only limited solubility, aqueous solutions of the compound (or of OsO₄) can be rapidly prepared by dissolution in a minimal amount of dimethylformamide and subsequent dilution with distilled water or buffer. Although stable in the solid state, the complex in solution undergoes partial dissociation releasing OsO₄, and the odor of OsO₄ becomes apparent.Such solutions of osmeth are (0.25%) considerably less concentrated with respect to OsO₄ than solutions (1–2%) ordinarily employed for ultrastructural preservation or in cytochemical studies. Osmeth has limited value for postosmication after glutaraldehyde fixation because the generation (release) of OsO₄ appears to be slow. Adequate osmication of tissue blocks exists only at the surface, but effective osmication can be achieved throughout tissue sections. In cytochemical reactions resulting in the formation of osmium blacks, the osmeth solutions are as effective as OsO₄ solutions of equivalent concentrations. Our findings indicate that OsO₄ solutions of less than 1% may be satisfactorily utilized in many cytochemical studies.Osmeth is safer and more convenient to handle than OsO₄ because small amounts may be solubilized as needed. It should be considered as a substitute for OsO₄ in ultrastructural cytochemistry.This investigation was supported by NIH research grant number DE 02668 from the National Institute of Dental Research and by NIH grant number RR 05333 from the Division of Research Facilities and ResourcesVisiting Professor, Dental Research Center, University of North Carolina at Chapel Hill, Jan.-May, 1975. Supported in part by USPHS Grant HD 09209     

Characterization of a monoclinic crystal form of an enzyme of steroid metabolism, Δ5-3-ketosteroid isomerase

A monoclinic crystal form ($\text{P2}{}_1\text{, a = 140.4}\text{A}\text{?}\text{, b = 85.0}\text{A}\text{?}\text{, c = 94.5}\text{A}\text{?}$, β= 130.1 °) of Δ⁵-3-ketosteroid isomerase from Pseudomonas testosteroni (EC 5.3.3.1), grown at pH 7.0, has been characterized. Crystal-density measurements show that the asymmetric unit contains 12 protomers (M_r = 13,394).     

Fitness of Karyotypes in DROSOPHILA PSEUDOOBSCURA

In the dynamics of the survival of chromosomal polymorphism selection may be operating at the genic level, at the chromosomal level or at the supergene level. Tests designed to distinguish between these levels were run on Drosophila pseudoobscura. There was no evidence for heterosis, a necessary requirement for gene-determined chromosomal polymorphism. A strong chromosmal selection was observed. No evidence was found for the presence within one locality of more than a single superallele for each supergene (= gene order). These results are compared to those found by others.     

Eimeria acervulina,E. brunetti,E. maxima, and E. necatrix: Low doses of oocysts to immunize young chickens

Resistance to reinfection varied with the species of Eimeria and with the number of oocysts in the inoculum. Chickens immunized with doses of 20,000 and 80,000 oocysts of E. acervulina, 312 and 1250 oocysts of E. brunetti or E. necatrix, or 1250 and 5000 oocysts of E. maxima at 2 and 4 weeks of age, respectively, were almost completely immune to a challenge dose at 6 weeks of age. Resistance was slightly less in chickens immunized with 1250 and 5000 oocysts of E. acervulina or 312 and 1250 oocysts of E. maxima. Birds given three immunizing infections of 1250, 5000, and 20,000 oocysts of E. maxima were completely immune 8 weeks after the last dose. Resistance was slightly less in birds immunized with similar doses of E. brunetti or E. necatrix. Doses of 20,000, 80,000, and 320,000 oocysts appeared necessary to confer a high level of immunity to E. acervulina. More than three low doses of oocysts appear necessary to induce a complete and enduring immunity against a high challenge for E. acervulina, E. brunetti, and E. necatrix. Higher immunizing doses would not be satisfactory due to the pathogenic effects of the coccidia after the initial infection.     

Further Studies on the Ribosomal RNA Cistrons of SCIARA COPROPHILA (Diptera)

Additional experiments with homologous as well as heterologous hybridization confirmed our previous finding in Sciara coprophila that XX females have nearly twice the number of ribosomal RNA cistrons as XO males. A comparison between two different X' chromosomes revealed that only the one carrying the irradiation-induced Wavy mutation has a deletion of 70% of its ribosomal RNA cistrons as compared to the standard X. The deletion is relatively stable, and the remaining ribosomal RNA cistrons donot appear to undergo disproportionate replication or magnification as in Drosophila. Homologous hybridization experiments revealed an unusually low reiteration of ribosomal RNA cistrons in this fly, 45 gene copies per X chromosome. The question is raised as to whether such a low number of cistrons may be related to the unusual nucleolar condition encountered in the Sciaridae.     

Modulation by prostaglandins of contractions in guinea-pig ileum

A high concentration of indomethacin (40μg/ml) substantially reduced contractions of guinea-pig isolated ileum in Krebs solution to nerve stimulation with electrical pulses or nicotine. Responses to acetylcholine and histamine were also inhibited, but to a smaller extent. Low concentrations of prostaglandin E₂ (2 or 4ng/ml) mainly restored all the excitatory responses. Using a modified bathing solution (lacking in phosphate and with some other changes) indomethacin 0.36μg/ml selectively inhibited nerve-mediated contractions. The results explain differences in various reports, and support the possibility that prostaglandins modulate the response to cholinergic nerve activity.     

Optimising Immunogenicity with Viral Vectors: Mixing MVA and HAdV-5 Expressing the Mycobacterial Antigen Ag85A in a Single Injection

The Bacillus Calmette - Guerin (BCG) vaccine provides a critical but limited defense against Mycobacterium tuberculosis (M.tb). More than 60 years after the widespread introduction of BCG, there is an urgent need for a better vaccine. A large body of pre-clinical research continues to support ongoing clinical trials to assess whether viral vectors expressing M.tb antigens that are shared by BCG and M.tb, can be used alongside BCG to enhance protection. A major focus involves using multiple unique viral vectors to limit anti-vector immunity and thereby enhance responses to the insert antigen delivered. The successful introduction of viral vector vaccines to target M.tb and other pathogens will be reliant on reducing the costs when using multiple vectors and inhibiting the development of unwanted anti-vector responses that interfere with the response to insert antigen. This study examines methods to reduce the logistical costs of vaccination by mixing different viral vectors that share the same insert antigen in one vaccine; and whether combining different viral vectors reduces anti-vector immunity to improve immunogenicity to the insert antigen. Here we show that a homologous prime-boost regimen with a mixture of MVA (Modified Vaccinia virus Ankara) and Ad5 (human adenovirus type 5) vectors both expressing Ag85A in a single vaccine preparation is able to reduce anti-vector immunity, compared with a homologous prime-boost regimen with either vector alone. However, the level of immunogenicity induced by the homologous mixture remained comparable to that induced with single viral vectors and was less immunogenic than a heterologous Ad5 prime-MVA-boost regimen. These findings advance the understanding of how anti-vector immunity maybe reduced in viral vector vaccination regimens. Furthermore, an insight is provided to the impact on vaccine immunogenicity from altering vaccination methods to reduce the logistical demands of using separate vaccine preparations in the field.     

Nicotinic Receptor-Mediated Release of Noradrenaline in the Human Neuroblastoma SH-SY5Y

Abstract: Dimethylphenylpiperazinium iodide (a nicotinic agonist) evokes noradrenaline release from human neuroblastoma SH-SY5Y cells that have been pretreated with 12- O -tetradecanoylphorbol 13-acetate for 8 min. This effect of dimethylphenylpiperazinium iodide was inhibited by 1 μ M mecamylamine but not by 1 μ M atropine, which suggests that SH-SY5Y cells express nicotinic receptors coupled to the release of noradrenaline. Dimethylphenylpiperazinium iodide-evoked release was enhanced by 5 μ M Bay K 8644 (an L-type calcium agonist) and inhibited by 1 μ M nifedipine. Dimethylphenylpiperazinium iodide depolarised SH-SY5Y cells and enhanced the level of intracellular calcium in cells loaded with fura 2. The effects of dimethylphenylpiperazinium iodide on noradrenaline release, depolarisation, and intracellular calcium levels were all inhibited by 1 μ M desmethylimipramine. The results of this study show that nicotinic receptors in SH-SY5Y cells stimulate noradrenaline release by activation of L-type calcium channels.