Dual regulation of adenylate cyclase and guanylate cyclase: alpha 2-adrenergic signal transduction in adrenocortical carcinoma cells

Isolated adrenocortical carcinoma cells of rat contain alpha 2- and beta-adrenergic receptors. When these cells are incubated with alpha 2-adrenergic agonists, there is a concentration-dependent increase of cyclic GMP that is blocked by the alpha 2-adrenergic antagonist yohimbine but not by the beta-antagonist propranolol. Concomitantly, both p-aminoclonidine (20 microM) and clonidine (100 microM), the alpha 2-adrenergic agonists, stimulate membrane guanylate cyclase activity. In calcium free medium there is no alpha 2-agonist-dependent increase in cyclic GMP. Isoproterenol, a beta-agonist, and forskolin cause an increase in cyclic AMP but not cyclic GMP. The cyclic AMP increase induced by isoproterenol is blocked by propranolol but not by yohimbine. Isoproterenol- and forskolin-dependent increases in cyclic AMP are inhibited by p-aminoclonidine and the inhibition is relieved by yohimbine. These results indicate a dual regulation of guanylate cyclase and adenylate cyclase by the alpha 2-receptor signal: guanylate cyclase is coupled to the receptor in a positive fashion, whereas adenylate cyclase is coupled in a negative fashion. Calcium is obligatory in the cyclic GMP-mediated response.     

Regeneration of plantlets from the callus of stem segments of adult plants of Ficus religiosa L.

Stem segments of adult plants of Ficus religiosa L. cultured on MS medium containing 1.0 mg/l 2,4-D produced callus. Shoots were regenerated when the induced calli were transferred to medium supplemented with 0.05 to 2.0 mg/l BAP. Callus derived shoots produced roots and developed into plantlets when transferred to medium supplemented with 1.0 mg/l NAA.Abbreviations MS Murashige and Skoog (1962) - BAP 6-benzylaminopurine - NAA naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid     

Plant regeneration from cotyledon protoplasts of Brassica carinata

Protoplasts isolated from cotyledons of Brassica carinata, underwent sustained division when cultured at 5.0 × 10⁴ ml^-1 in modified 8p medium (KM8P) with 1.0% (w/v) Seaplaque agarose. Cell colonies produced callus when agarose droplets, in which the protoplasts were embedded, were transferred to K8 medium with 0.6% (w/v) Sigma Type I or Type VII agarose at day 16, giving a plating efficiency of 1.6%. Seventy percent of the protoplast derived-tissues produced shoot buds after subculture to MS medium containing 3.0% (w/v) sucrose, 1.125 mgl^-1 BAP, 0.035 mgl^-1 GA and 0.6% (w/v) Type I agarose, resulting in shoot formation from 1.1% of the protoplasts originally plated. Protoplast-derived colonies transferred to hormone-free MS medium with 1.0% (w/v) sucrose and 0.6% (w/v) Type I agarose produced roots. The latter gave rise to shoots after excision from the parent callus and culture on MS medium with 3.0% sucrose, 0.225 mgl^-1 BAP, and 0.6% (w/v) Type I agarose. Shoots regenerated directly from protoplast-derived calli, or indirectly from roots, developed prolific root systems when placed on hormone-free MS medium with 1.0% (w/v) sucrose and 0.6% (w/v) Type I agarose.Abbreviations BAP 6-benzylaminopurine - CH casein hydrolysate - 2,4-D 2,4-dichlorophenoxyacetic acid - GA gibberellic acid - K kinetin - NAA -naphthaleneacetic acid - MES 2(N-morpholino)ethanesulphonic acid, 2,iP-6(,-dimethylallyamino) purine - IAA indole-3-acetic acid - Z zeatin - ZR zeatin riboside     

Large scale production of recombinant mouse and rat growth hormone by fed-batch GS-NSO cell cultures

Investigations of biological effects of prolonged elevation of growth hormone in animals such as mice and rats require large amounts of mouse and rat growth hormone (GH) materials. As an alternative to scarce and expensive pituitary derived materials, both mouse and rat GH were expressed in NSO murine myeloma cells transfected with a vector containing the glutamine synthetase (GS) gene and two copies of mouse or rat GH cDNA. For optimal expression, the mouse GH vector also contained sequences for targeting integration by homologous recombination. Fed-batch culture processes for such clones were developed using a serum-free, glutamine-free medium and scaled up to 250 L production scale reactors. Concentrated solutions of proteins, amino acids and glucose were fed periodically to extend cell growth and culture lifetime, which led to an increase in the maximum viable cell concentration to 3.5×10⁹ cells/L and an up to 10 fold increase in final mouse and rat rGH titers in comparison with batch cultures. For successful scale up, similar culture environmental conditions were maintained at different scales, and specific issues in large scale reactors such as balancing oxygen supply and carbon dioxide removal, were addressed. Very similar cell growth and protein productivity were obtained in the fed-batch cultures at different scales and in different production runs. The final mouse and rat rGH titers were approximately 580 and 240 mg/L, respectively. During fed-batch cultures, the cell growth stage transition was accompanied by a change in cellular metabolism. The specific glucose consumption rate decreased significantly after the transition from the growth to stationary stage, while lactate was produced in the exponential growth stage and became consumed in the stationary stage. This was roughly coincident with the beginning of ammonia and glutamate accumulation at the entry of cells into the stationary stage as the result of a reduced glutamine consumption and periodic nutrient additions.     

[2-3H]ATP synthesis and 3H NMR spectroscopy of enzyme-nucleotide complexes: ADP and ADP.Vi bound to myosin subfragment 1

Summary The synthesis of [2-³H]ATP with specific activity high enough to use for ³H NMR spectroscopy at micromolar concentrations was accomplished by tritiodehalogenation of 2-Br-ATP. ATP with greater than 80% substitution at the 2-position and negligible tritium levels at other positions had a single ³H NMR peak at 8.20 ppm in 1D spectra obtained at 533 MHz. This result enables the application of tritium NMR spectroscopy to ATP utilizing enzymes.The proteolytic fragment of skeletal muscle myosin, called S1, consists of a heavy chain (95 kDa) and one alkali light chain (16 or 21 kDa) complex that retains myosin ATPase activity. In the presence of Mg²⁺, S1 converts [2-³H]ATP to [2-³H]ADP and the complex S1.Mg[2-³H]ADP has ADP bound in the active site. At 0°C, 1D ³H NMR spectra of S1.Mg[2-³H]ADP have two broadened peaks shifted 0.55 and 0.90 ppm upfield from the peak due to free [2-³H]ADP. Spectra with good signal-to-noise for 0.10 mM S1.Mg[2-³H]ADP were obtained in 180 min. The magnitude of the chemical shift caused by binding is consistent with the presence of an aromatic side chain being in the active site. Spectra were the same for S1 with either of the alkali light chains present, suggesting that the alkali light chains do not interact differently with the active site. The two broad peaks appear to be due to the two conformations of S1 that have been observed previously by other techniques. Raising the temperature to 20 °C causes small changes in the chemical shifts, narrows the peak widths from 150 to 80 Hz, and increases the relative area under the more upfield peak. Addition of orthovanadate (V_i) to produce S1.Mg[2-³H]ADP.V_i shifts both peaks slightly more upfield without chaning their widths or relative areas.     

Human and animal mesenchymal progenitor cells from bone marrow: Identification of serum for optimal selection and proliferation

Summary An undifferentiated subset of cells within the stromal cell population of bone marrow in postnatal mammals retains the capacity to differentiate along osteogenic, adipogenic, fibroblastic, and chondrogenic lines. These cells, which are referred to as mesenchymal stem cells (MSCs), can be maintainedin vitro and expanded in number through a process of subculturing. MSCs are maintained in culture in medium supplemented with 10% fetal bovine serum (FBS). It is believed that certain, as yet unidentified, serum components play critical roles in the attachment and proliferation of MSCs. Commercially available FBS is poorly characterized and may vary in composition and quality from lot to lot. This study describes a method for the selection of lots of FBS that best support maintenance of the undifferentiated state, mitotic expansion of MSCsin vitro, and retention of multilineage developmental potential in response to appropriate cues.     

Effect of dihydroergotoxine, a cerebral vasodilator, on cognitive deficits induced by prenatal undernutrition and environmental impoverishment in young rats

The study was conducted on 64 Charles Foster strain albino rats, which were equally distributed into 8 evenly matched groups, following a 2 x 2 x 2 factorial design, by varying three independent factors at two levels: nutrition--normal and undernutrition; environment--enrichment and impoverishment, and drug treatment--vehicle and dihydroergotoxine (3 mg/kg, i.p.). Prenatal undernutrition was induced by restricting the mother's food intake. The environmental enrichment/impoverishment and the vehicle/dihydroergotoxine treatments were given during the postweaning period of the pups. The rats were subjected to original and subsequent reversal brightness discrimination learning tests in a single unit T-maze at 8-9 weeks of age. Thereafter, the animals were tested for passive avoidance learning. The results indicate that undernutrition caused significant original and reversal discrimination learning, deficits whereas environmental deprivation attenuated only the original discrimination learning performance. Dihydroergotoxine treatment facilitated the learning performance of rats in both the original and reversal learning tests. Nutritional, environmental and dihydroergotoxine treatments had no effect on the retention of the passive avoidance learning, both at 24 hr and 1 week intervals. Dihydroergotoxine treatment attenuated the learning deficits induced by prenatal undernutrition. The results indicate that dihydroergotoxine is not likely to be useful in cognitive deficits, induced by malnutrition, though it facilitated learning acquisition, since it had no effect on retention.