Recombinant human prorenin from CHO cells: Expression and purification

The gene for human preprorenin was obtained from total RNA prepared from primary human chorion cells. An expression vector was constructed containing an SV40 early promoter, a human preprorenin cDNA, bovine growth hormone poly-A addition signal, and a dihydrofolate reductase (dhfr) expression cassette. This vector was inserted into the DXB-11 Chinese hamster ovary (CHO) cell line. The recombinant protein was exported by CHO cells into the tissue culture media. At harvest the prorenin levels ranged from 1–5 mg/L. For prorenin isolation the cell culture supernatants were processed by filtration, concentration, dialysis, and batch extraction. Preparative-scale isolation of prorenin was accomplished using blue-dye chromatography and size-exclusion chromatography. The isolated prorenin yielded a single SDS-gel band with Mr 40,000. The proprotein was characterized with respect to N-terminal sequence and N-linked sugar composition. Trypsin-activated renin prepared from the proprotein was characterized with respect to N-terminal sequence andpH-activity profile. Enzyme activity was measured with a newly developed fluorogenic peptide substrate containing the P₆-P₃ sequence of human angiotensinogen.     

Dose- and time-dependent hippocampal cholinergic lesions induced by ethylcholine mustard aziridinium ion: Effects of nerve growth factor,GM1 ganglioside,and vitamin E

Ethylcholine mustard aziridinium ion (ECMA) was infused intracerebroventricularly (icv) to rats followed by measurement of two markers of presynaptic cholinergic neurons, choline acetyltransferase (ChAT) activity and high affinity choline transport (HAChT), in the hippocampus and cortex. Bilateral icv administration of 1, 2, or 3 nmol of ECMA per side produced dose-dependent reductions in each marker in the hippocampus, but not in the cortex, one week after treatment. Reductions of 52% and 46% for ChAT activity and HAChT, respectively, were produced in the hippocampus by 3 nmol ECMA. Measurement of these two markers at different times after icv infusion of 2 nmol ECMA/ventricle revealed that the activity of ChAT was reduced to a greater extent than was HAChT in the hippocampus 1 day and 1, 2, 4, and 6 weeks after treatment. The maximal reductions of ChAT activity and HAChT (61% and 53%, respectively) were reached between 1 and 2 weeks after ECMA administration. There was no evidence of regeneration of either marker at 4 or 6 weeks posttreatment. HAChT and ChAT activity in the cortex were not altered at any of the posttreatment times examined.ECMA-induced deficits in hippocampal ChAT activity and HAChT were not counteracted by the following treatments: (i) daily administration of GM₁ ganglioside (10 mg/kg, intraperitoneally (ip)) from the day prior to infusion of ECMA until 2 weeks later; (ii) daily administration of GM₁ ganglioside between 2 and 6 weeks after infusion of ECMA; and (iii) icv administration of nerve growth factor (NGF) twice per week for 2 weeks after ECMA treatment. Since similar treatments with NGF and GM₁ ganglioside ameliorate lesions induced by other methods, these results indicate that the mechanism of lesion formation and the surviving cellular components influence the functional effects of neurotrophic factors. In contrast to the above results, treatment with vitamin E significantly attenuated ECMA-induced deficits of ChAT activity and HAChT. Further studies of the effects of vitamin E on the development of ECMA-induced deficits may help to elucidate the mechanism action of ECMA.     

Somatic embryogenesis and plant regeneration from immature embryos of western larch

Somatic embryogenesis was initiated from immature embryos of western larch (Larix occidentalis Nutt.) on media containing 2,4-dichlorophenoxyacetic acid and N6- benzyladenine. The effects of explant type and ammonium nitrate and glutamine concentrations on initiation were tested. Although 21–93% of explants rendered cultures in various experiments, only 3% yielded sustainable embryogenic lines. Excised embryos at the early cotyledonary stage were optimal for initiation. Maturation of somatic embryos was promoted by abscisic acid. Response to abscisic acid concentrations and duration of exposure to abscisic acid varied with genotype. Maximal results were obtained with 0.025 M abscisic acid for 1 to 2 weeks followed by individual culture on medium without growth regulators. Mature somatic embryos developed into shoots with roots. Plantlets have been established in peat.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BA N⁶-benzyladenine - IBA indole-3-butyric acid - ABA abscisic acid     

The cellular and molecular regulation of 1,25(OH)2D3 production

The synthesis of 1,25(OH)₂D₃ is a critical control point in the regulation of calcium metabolism, and possibly in the growth and differentiation of a number of cell types. This paper reviews our current understanding of the regulation of this process at the cellular and molecular levels, with the emphasis on the mechanisms of feedback control 1,25(OH)₂D₃ itself, control of parathyroid hormone, the roles of cyclic AMP dependent protein kinase and protein kinase C, and the interaction between the various intracellular regulators of 1,25(OH)₂D₃ production.     

Implications of the human genome initiative for the primary care physician

... Despite the need for physicians to be knowledgeable about and open to advances in genetic technology, little is known about the level of preparedness of primary care physicians to offer new genetic tests. Evidence suggests that several barriers exist to physicians adopting genetic tests. These include lack of knowledge, inability to interpret probabilistic information, low tolerance for uncertainty, negative attitudes about their responsibility for genetic counseling and testing, lack of confidence in their clinical skills, and unfamiliarity with ethical issues raised by testing. This paper will explore some of these barriers in further depth, discuss the ethical impact of physician unpreparedness on both patient care and the diffusion of genetic tests, and describe a study that is currently underway to investigate some of these issues.     

Leaf growth of Betula and Acer in simulated shadelight

Summary Leaves of birch (Betula pendula Roth) and sycamore (Acer pseudoplatanus L.) were initiated and grown either in a simulated shadelight (80 mol m^-2 s^-1, R/FR ratio 0.28)/dark photoenvironment or a white light (250 mol m^-2 s^-1, R/FR>1)/dark photoenvironment. Until the leaves were more than 50% expanded, growth rates (measured every 24 h) were the same for both species in both environments. After this time, growth rate slowed and this correlated well with a decrease in wall extensibility (WEX). Birch leaves in shadelight showed reduced surface acidification and were the first to show reduced growth. WEX under these conditions was particularly low.Daily patterns of leaf growth of the two species were very different. Sycamore leaves showed a slightly higher growth rate in the dark than in shadelight, while birch leaves grew more rapidly in shadelight than in the dark. Limitation of growth of sycamore leaves in light may be explained by a very high yield threshold turgor for growth (Y). The daily pattern of leaf growth shown by birch is more difficult to explain but the importance of a possible limitation of growth by solute availability and a diurnal variation in Y are discussed.     

Hormonal regulation of fat esterification & oxidation in hepatocytes from fed rats: modulation by 3-mercaptopicolinate

3-Mercaptopicolinate, an inhibitor of phosphoenolpyruvate carboxykinase (PEPCK), decreased esterification of [1-14C] oleate and [1-14C] myristate in hepatocytes from fed rats. In the absence of 3-mercaptopicolinate, adrenaline, noradrenaline, vasopressin or angiotensin II increased esterification to triacylglycerol of [1-14C] oleate but not [1-14C] myristate. Cyclic AMP decreased esterification of both oleate and myristate. In the presence of 3-mercaptopicolinate, stimulation of oleate esterification by the catecholamines, vasopressin or angiotensin II was increased, and stimulatory effects of these hormones on myristate esterification were observed. Adrenaline, noradrenaline, vasopressin or angiotensin II increased 14CO2 production from both [1-14C] oleate and [1-14C] myristate but the degree of stimulation was similar in the absence or presence of 3-mercaptopicolinate. The results indicate a role for the catecholamines and angiotensin II in the regulation of liver fat metabolism and emphasize the potential importance of changes in activity of PEPCK as determinants of hepatic carbon flux.     

Ketone-body metabolism after partial hepatectomy in the rat.

Fed or 24 h-starved rats were subjected to two-thirds partial hepatectomy or sham-operation and subsequently starved for 4, 14 or 24 h. Despite high plasma fatty acid concentrations, the partially hepatectomized rats failed to respond to post-operative starvation with increased blood and liver ketone-body concentrations or to maintain the high ketone-body concentrations associated with pre-operative starvation. Hypoglycaemia and hyperlactaemia were observed within 30 min of functional hepatectomy, but not partial hepatectomy, of 24 h-starved rats, and, even after a further 24 h starvation of partially hepatectomized rats, blood glucose concentrations were only slightly decreased. The results are discussed with reference to fat oxidation and gluconeogenesis in the liver remaining after partial hepatectomy.