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First, we describe a preparation of sealed unilamellar lipid vesicles. When this preparation was subjected to sucrose density gradient centrifugation, two rather uniform fractions emerged, one consisting of lighter lipid-rich vesicles with average diameters ranging over 150–200 nm (fraction I), the other consisting of heavier vesicles with average diameters ranging over 30–70 nm (fraction II). When the lipid mixture containing dimyristoylglycero-phosphocholine, cholesterol, dipalmitoylglycerophosphoserine and dipalmitoyglycerophosphoethanolamine at molar ratios of 54:35:10:1 was reconstituted with α- and γ-subunits of G0-proteins purified to homogeneity from bovine brain, the lipid-rich lighter vesicle fraction I took up these subunits nearly exclusively. Whereas, when a β1-adrenoceptor preparation purified from turkey erythrocyte membranes was reconstituted, it was found nearly completely in the smaller heavier vesicle fraction II where it was incorporated inside-out. On co-reconstitution of either αo or alone with β1-adrenoceptors, some of these subunits appear together with β1-adrenoceptors in the small vesicle fraction II, but much more αo was bound to the receptor in the presence of βγ-subunits. The observations reported are novel and surprising in several respects: firstly, they suggest that βγ-subunits can bind to the non-activated β1-receptor where they may serve as an anchor for α-subunits. Secondly, the binding of αo- and βγ-subunits to the β1-adrenoceptors enhances the basal GTPase activity of αo. Thirdly, since the binding domains of the β1-adrenoceptor for G-proteins were facing outwards in our sealed vesicle preparations, it follows that interactions of G-proteins with the β-receptor can occur at the aqueous membrane interface as was postulated originally by M. Chabre [Trends Biochem. Sci. 12, 213–215 (1987)] for the transducin-rhodopsin interactions. Finally, the binding of Go-subunits from bovine brain to a β1-adrenoceptor from turkey erythrocytes was not expected, since these polypeptides are not likely to be physiological partners.

Methods of analysis for products of modern biotechnology are required for national and international trade in seeds, grain and food in order to meet the labeling or import/export requirements of different nations and trading blocks. Although many methods were developed by the originators of transgenic events, governments, universities, and testing laboratories, trade is less complicated if there exists a set of international consensus-derived analytical standards. In any analytical situation, multiple methods may exist for testing for the same analyte. These methods may be supported by regional preferences and regulatory requirements. However, tests need to be sensitive enough to determine low levels of these traits in commodity grain for regulatory purposes and also to indicate purity of seeds containing these traits. The International Organization for Standardization (ISO) and its European counterpart have worked to produce a suite of standards through open, balanced and consensus-driven processes. Presently, these standards are approaching the time for their first review. In fact, ISO 21572, the "protein standard" has already been circulated for systematic review. In order to expedite the review and revision of the nucleic acid standards an ISO Technical Specification (ISO/TS 21098) was drafted to set the criteria for the inclusion of precision data from collaborative studies into the annexes of these standards.

Glucagon and prostaglandin E 1 stimulate adenylate cyclase in Madin-Darby canine kidney cells with an approximate EC 50 of 3 ∗ 10 −8 and 1 ∗ 10 −7 M respectively. The rise in cAMP is accompanied by a transient rise in intracellular Ca ++ measured with the fluorescent calcium indicator Indo-1. A comparable increase in intracellular Ca 2+ without a rise in cAMP occurs with the cholinergic agonist carbamylcholine. Stimulation of adenylate cyclase by the β-adrenergic agonist isoproterenol or directly by forskolin has no effect on intracellular Ca ++ . By all criteria studied the rise in intracellular Ca ++ and the increase in cAMP are independent from each other.

Objective: Apolipoprotein E (APOE) E4, apolipoprotein B-100 (APOB) Q3611 allele, the angiotensin converting enzyme (ACE) deletion (D) allele and glycoprotein IIIa (GP3A) P33 mutant allele are reported to predispose to early-onset coronary heart disease (CHD). These associations were not all confirmed in more recent studies. To determine the impact of these alleles on CHD, we examined the prevalence of these mutations in patients presenting with early-onset CHD and compared them to those manifesting CHD later in life. The delayed-onset was considered a sign of longevity and would serve as a comparative group to assess prevalence of the biochemical and genetic risk factors. Methods: 300 patients with a history of myocardial infarction or angina pectoris and angiographically documented CHD were studied. Patients were divided into two groups: group 1 (G1 = 150 patients) presenting with these findings under the age of 50 years; while group 2 (G2 = 150 patients) were patients presenting for the first time over the age of 65 years. Prevalence of the alleles of APOE, APOB, ACE and GP3A was assessed by molecular analysis. An association of any of these genotypes with early onset CHD could lead to a higher prevalence in the younger age group. Results and Conclusions: None of the suspected alleles namely APOB Q3611 [G1: 10.7% vs. G2: 9.0%, p = 0.57], ACE D (G1: 52.0% vs. G2: 49.7%, p = 0.57), or the GP3A P33 (G1: 17.3% vs. G2: 15.7%; p = 0.58) showed any significant difference between the two groups. Subjects with APOE E4 were more frequent in the younger age group (G1: 18.3% vs. G2: 13.7%; p = 0.047), while APOE E2 was more frequent in G2 (G2: 10.0% vs. G1: 2.7%; p = 0.0002). Multivariate analysis showed an odds ratio of APOE E2 allele in G1 of 0.27 with a confidence interval of 0.10–0.73.

Polyunsaturated fatty acids (PUFA) have a selective cytotoxic/cytostatic effect on a number of tumor cell lines in culture. Although this process may be enhanced by the addition of iron there is a minimum level of PUFA necessary for potentiation of cell death. Vitamin E blocks PUFA cytotoxicity when added up to 5 days after fatty acid administration. Levels of thio-barbiturate reactive material (TBARM) in the medium rise in parallel with cell death. However, they are not affected by small alterations in temperature or oxygen tension. Incubating cells with PUFA causes marked alterations in the fatty acid patterns of both neutral and phospholipid fractions. Membrane fluidity is increased and the activity of membrane-bound receptors may be influenced directly or through the actions of eicosanoids derived from the exogenous fatty acid. PUFA may be an effective way of influencing tumor growth and a safe approach for the management of human cancer.

New fluorescent glucagon derivatives were synthesized by converting tryptophan25 to 2-thiol-tryptophan with the consequent use of thiol specific fluorescent reagents. All derivatives retained the ability to bind tightly to rat liver membranes and rat hepatocytes in primary culture and to activate adenylate cyclase as potently as native glucagon. Thus these derivatives are full agonists. From experiments with monolayer cultured hepatocytes and 125I-glucagon at elevated temperatures it was assumed that the ligand was internalised at this temperature since some of the specifically bound ligand could no longer be washed off with acid. This was confirmed in experiments where monolayer cultures of hepatocytes were incubated with the fluorescein-labelled derivates of glucagon, thus allowing the study of the distribution of glucagon specifically bound on the cell surface using video intensification microscopic techniques. In keeping with autoradiographic studies using radiolabelled glucagon, or electron microscope studies using ferritin labelled glucagon, we could now show using fluorescently labelled glucagon derivatives and video intensification microscopy that at lower temperatures the bound ligand was distributed all over the cell surface. Whereas, at the higher temperature, ligand derived fluorescence could only be detected in mobile intracellular vesicles following internalisation and removal from the cell surface.

When monolayer cultured hepatocytes were incubated with 1 nM [125I]glucagon at 30 degrees C, equilibrium was reached after 10 min, whereas at 4 degrees C, equilibrium was reached after 60 min. At the higher temperature, 11.2% of the bound ligand was broken down after 60 min, at the lower temperature, the amount of degradation was negligible. At 30 degrees C, acid-washing did not remove specifically bound ligand; thus, it was assumed that the ligand was internalised at this temperature, since some of the specifically bound ligand could be washed off at lower temperatures. This was confirmed in experiments when monolayer cultures of hepatocytes were incubated with fluorescein-labelled derivatives of glucagon. The distribution of specific binding on the cell surface was studied at both 30 and 4 degrees C using video intensification microscopic techniques. In keeping with studies using radiolabelled glucagon, more fluorescence was detected following incubation at 4 degrees C than at 30 degrees C and it could be removed by washing the cells. Video intensification microscopy indicated that at the lower temperature, the bound ligand was distributed all over the cell surface. At the higher temperature, ligand-derived fluorescence could only be detected in mobile intracellular vesicles.

Anti-peptide antibodies, raised against the N-terminal sequence (amino acids 2-10) of the turkey beta 1-adrenoceptor [Yarden et al., Proc. Natl. Acad. Sci. USA (1986) 83, 6795-6799] recognized the 50 kDa- but not the 40 kDa-form of the receptor, thus confirming the previous assumption that the N-terminus of the 50 kDa form is lost during its conversion to the 40 kDa-form [Jür beta, R., Hekman, M.Helmreich, E.J.M. (1985) Biochemistry 24, 3349-3354]. By in situ proteolysis small amounts of receptor fragments were formed, which could be recognized by the N-terminus specific antibody. Therefore, although the production of the stable 40 kDa receptor species by proteolytic removal of a portion of the N-terminal appears to be the predominant route, there exists an additional pathway of degradation which must involve the initial cleavage of the carboxyl terminal.

1. 1. Age-related changes in the activities of fatty acid synthetase and palmitoyl-CoA synthetase (EC 6.2.1.3) have been determined in rabbit brain from the foetal stage through to maturity. 2. 2. Fatty acid synthetase was most active in the soluble fraction of brain homogenates at 5 days of age, prior to the active phase of myelination. Palmitic acid was the major fatty acid synthesised throughout development. 3. 3. Palmitoyl-CoA synthetase had a constant specific activity in the full homogenate, but in the microsomal fraction reached a maximum specific activity at 15–20 days of age. The specific activity was higher than in the mitochondrial fraction which declined from birth. Most of the palmitoyl-CoA synthetase activity was present in the fraction containing cell membranes plus nuclei. 4. 4. From a comparison of the total activities of the enzymes involved in the metabolism of fatty acids in the brain, de novo fatty acid synthesis may be rate limiting compared with the esterification of synthesised fatty acids, but not in the further transformations of the synthesised fatty acids.

The synthesis and properties of a fluorescent derivative of the hydrophilic beta-adrenergic antagonist CGP-12177 are described. The fluorescence of the NBD derivative of CGP-12177 (CGP-NBD) is extremely sensitive to its environment, the quantum yield increasing 23-fold upon transfer from water to acetonitrile. This property of CGP-NBD was taken into account and a procedure was developed using quantitative chloroform extraction of ligand for the measurement of CGP-NBD bound specifically to beta-receptors on A431.E3 membranes. The fluorescent NBD-derivative of CGP-12177 bound strongly and specifically to A431 cells, a KD of 3.9 x 10(-10) M being measured; the specific binding represented 63% of the total binding at a concentration of 1 x 10(-8) M (256 x KD). A431.E3 cells were used for the binding studies since they gave consistently higher receptor numbers when compared with the native strain. A maximal number of 47,000 sites/cell and a KD of 100 pM were measured with CGP-12177 on adhered cells. The receptor number was strongly dependent upon cell density with only 3000 sites/cell being measured in suspension at confluence.

The synthesis of monofluorescein, monorhodamine, and mono-4-nitrobenz-2-oxa-1,3-diazole (NBD) derivatives of glucagon is reported. The fluorescent groups were introduced by converting tryptophan-25 to 2-thioltryptophan using thiol-specific fluorescent reagents. All derivatives retained the ability to activate adenylate cyclase when compared to glucagon and thus were considered full agonists. IC 50 values of 6.8·10 −9 , 1.7·10 −8 , 1.8·10 −8 and 5.4·10 −9 M were measured in rat liver membranes for NBD-, fluorescein-, rhodamine-Trp 25 -glucagon and native glucagon, respectively. From the IC 50 values K d values of 2.16·10 −9 , 4·10 −9 , 2·10 −9 and 1.72·10 −9 M were calculated for the binding of NBD-, fluorescein-, rhodamine-Trp 25 -glucagon and native glucagon, respectively. The highest quantum yield (0.18) of the monomer derivatives was obtained with fluorescein-Trp 25 -glucagon in phosphate-buffered saline (pH 7.4). Difluorescein-glucagon was also prepared by reacting the amino groups of histidine-1 and lysine-12 with fluorescein isothiocyanate and dimer derivatives were prepared using fluorescein-labelled 2-thiolTrp 25 -glucagon. Difluorescein-glucagon bound only weakly to glucagon receptors and displayed antagonist properties. The dimer derivative formed from two difluorescein-2-thiolTrp 25 -glucagon molecules had similar poor binding qualities, whereas the dimer formed from difluorescein-2-thiolTrp 25 -glucagon and 2-thiolTrp 25 -glucagon exhibited, at low concentrations, properties similar to monofluorescein-glucagon. Both dimer derivatives were only sparingly soluble in aqueous medium. Specific binding of fluorescein-Trp 25 -glucagon and difluorescein-glucagon to rat hepatocytes was followed using flow cytometry.