A survey of first-year biology student opinions regarding live lectures and recorded lectures as learning tools.

A cohort of first-year biology students was surveyed regarding their opinions and viewing habits for live and recorded lectures. Most respondents (87%) attended live lectures as a rule (attenders), with 66% attending more than two-thirds of the lectures. In contrast, only 52% accessed recordings and only 13% viewed more than two-thirds of the available recordings. Respondents regarded lectures as efficient for information delivery (75%), and 89% enjoyed live lectures because they were useful for learning (89%), understanding coursework (94%), and keeping up with the subject (93%). Lecture enjoyment was driven less by entertainment (34%) or interaction with the lecturers (47%), although most students preferred an entertaining lecturer to a factual expert (72%). Exam marks were positively correlated with the number of lectures attended (P < 0.001) and negatively correlated with the number of recordings viewed (P < 0.05), although marks were similar for lecture attenders and nonattenders (P > 0.05). Lecture attenders mostly missed lectures to complete assessments during the same week (68%), whereas nonattenders were more likely to miss lectures due to outside commitments or preference for study from books or recorded lectures (P < 0.001). Recordings were used to replace missed lectures (64%), rather than for revision, and were viewed mostly alone (96%) in one sitting (65%). Only 22% of respondents agreed that some lectures could be replaced by recordings, but 59% agreed with having some videoconference lectures from experts on another campus. Overall, this cohort showed a clear preference for live lectures over recordings, with limited support for synchronous videoconference lectures.

Seasonal and age effects on energy requirements in domestic short-hair cats (Felis catus) in a temperate environment.

There is little information known about the energy requirements of cats in temperature climates. Energy requirement of domestic short-haired cats was determined using three groups of mixed gender - old kept outside (approximately 9.9 years of age; 4.8 kg; n = 9), young kept outside (approximately 3.1 years of age; 3.9 kg; n = 8) or young kept inside (approximately 3.1 years of age; 3.9 kg; n = 8). Cats were housed individually for 5 weeks during summer (18.5 ± 0.5 °C) and winter (8.5 ± 0.4 °C) and were fed a commercially available maintenance diet ad libitum. In both periods, energy expenditure was determined from the rates of (2) H and (18) O elimination for blood H2 O over a 12 day period, from a doubly labelled water bolus (2) H2 O (0.7 g/kg BW) and H2 (18) O (0.13 g/kg BW) administered intravenously. During the summer period, macronutrient digestibility was determined. Older cats had a reduction (p < 0.05) in apparent digestibility of dry matter (approximately 9%), energy (approximately 8%) and protein (6%). There was a significant effect of age and season on energy intake and energy expenditure. While lean mass was affected by age and season, there was no effect of age or season on energy expenditure when expressed as a proportion of lean mass. Possible seasonal differences in nutrient digestibility may explain these results.

Mapping of beta-adrenoceptor coupling domains to Gs-protein by site-specific synthetic peptides.

Peptides corresponding to the known sequence of turkey erythrocyte beta 1-adrenergic receptor were synthesized and the effects on receptor-mediated cyclase activation were measured. Peptides corresponding to the first and second intracellular loops (T61-71 and T138-159) inhibited at micromolar concentrations the hormone-dependent cyclase activation in turkey erythrocyte membranes. In contrast, the peptide corresponding to the C-terminal part of the third intracellular loop (T284-295) increased the cyclase activity in a hormone-independent manner. Peptides T338-353 and T2-10 and a number of synthetic peptides unrelated to the beta-adrenoceptor had no effect.

Immunologic mapping of the amino- and carboxy-termini of the turkey erythrocyte beta-adrenergic receptor: selective proteolysis of both domains.

Peptide-directed antibodies were used to map the N- and C-termini of the turkey erythrocyte beta-adrenergic receptor, the full length recombinant receptor expressed in Sf9 cells, and a mutant that terminates after residue 424 (T424). Both forms of the natural receptor (P40 and P50) were proteolytically clipped between residues 419 and 424. P40, but not P50, is also proteolyzed between residues 14 and 28. Truncation mutants, but not full length receptors, also display both large and small forms. The short form of T424 is formed by proteolysis after residue 14, but neither form is proteolyzed in the C-terminal region. The wild type recombinant receptor is not proteolyzed.

Identification of a Gs-protein coupling domain to the beta-adrenoceptor using site-specific synthetic peptides. Carboxyl terminus of Gs alpha is involved in coupling to beta-adrenoceptors.

Competition between Gs-protein and the synthetic peptide, GSA 379-394, derived from the carboxyl-terminal region of the alpha s-subunit, led to complete inhibition of receptor-mediated adenylate cyclase activation in turkey erythrocyte membranes. Related peptides corresponding to the homologous carboxyl-terminal region of alpha t-, alpha il- or alpha o-subunits did not interfere with beta-receptor-Gs coupling. The direct coupling between Gs and adenylate cyclase was not influenced by any of these peptides. These results emphasize the important role of the carboxyl-terminus of G-protein alpha-subunits for the specific recognition of their corresponding receptors and for signal transduction.

Unimpaired coupling of phosphorylated, desensitized beta-adrenoceptor to Gs in a reconstitution system.

Heterologous desensitization of turkey erythrocyte beta-adrenoceptors correlates with receptor phosphorylation and impaired receptor-Gs coupling, as assessed by fusion of purified desensitized receptors with X. laevis erythrocytes [(1984) Science 225, 837-840]. We have purified beta-receptors from desensitized and untreated turkey erythrocytes and have compared the abilities of these two receptors to couple with pure turkey erythrocyte Gs in a reconstituted system. Functional receptor-Gs coupling was assessed by measuring hormone-dependent Gs activation by GTP gamma S and GTPase activity. While in membranes prepared from desensitized cells, receptor-Gs coupling was clearly reduced, this effect was absent when coupling of purified desensitized receptor was measured. We conclude that covalent modification by phosphorylation does not fully explain the functional uncoupling at the membrane level.

Phosducin inhibits receptor phosphorylation by the beta-adrenergic receptor kinase in a PKA-regulated manner.

Homologous or receptor-specific desensitization of beta-adrenergic receptors is thought to be triggered by receptor phosphorylation mediated by the beta-adrenergic receptor kinases (beta ARK). Upon receptor activation, cytosolic beta ARK translocates to the membrane, probably by binding to G-protein beta gamma-subunits. Using the purified proteins reconstituted into phospholipid vesicles we show here that this binding process can be inhibited by phosducin, a cytosolic protein that has recently been described as a regulator of G-protein-mediated signalling. Phosducin appears to complete very effectively with beta ARK for the G-protein beta gamma-subunits. These inhibitory effects of phosducin on receptor phosphorylation are antagonized following phosphorylation of phosducin by protein kinase A. It is proposed that phosducin may act as a regulator of homologous beta-adrenergic receptor desensitization.

Purification and functional characterization of beta-adrenergic receptor kinase expressed in insect cells.

The beta-adrenergic receptor kinase mediates agonist-dependent phosphorylation of beta-adrenergic receptors, which is thought to represent the first step of homologous desensitization. We have expressed bovine and human beta ARK1 in Sf9 cells and purified them to apparent homogeneity in milligram quantities. The Km-values of the enzyme were 3.8 microM for rhodopsin and 22 microM for ATP; the Vmax-value was 9.9 mol phosphate/mol beta ARK/min. These data indicate that the two recombinant kinases were at least as active as preparations previously obtained from bovine brain. There were no differences in the functional activity of human and bovine beta ARK.

Purification and functional characterization of the human beta 2-adrenergic receptor produced in baculovirus-infected insect cells.

A human cDNA fragment bearing the complete coding region for the beta 2-adrenergic receptor was introduced into the genome of Autographa california nuclear polyhedrosis virus under the control of the polyhedrin promoter. Binding studies using [125I]iodocyanopindolol showed that Sf9 insect cells infected with the recombinant virus expressed approximately 1 x 10(6) beta 2-adrenergic receptors on their cell surface. Photoaffinity labeling of whole cells and membranes revealed a molecular weight of approximately 46,000 for the expressed receptor. The receptor produced in insect cells is glycosylated but the extent and pattern differ from that of the receptor from human tissue. The heterologously expressed receptor was purified by alprenolol affinity chromatography, and was able to activate isolated Gs-protein.

The hormonal regulation of adenylate cyclase.

The regulation of adenylate cyclase by hormones and by GTP regulatory proteins was investigated in native membrane systems and in systems reconstituted from purified components. These studies can be summarized as follows. The stimulatory beta 1-adrenoceptor catalyses the activation of a complex between the GTP stimulatory protein GS and the catalytic unit C. The agonist-receptor complex can activate a few cyclase units in native membrane systems as well as in reconstituted systems. GS from turkey erythrocytes is functionally different from rabbit liver GS, the latter being more amenable to activation by guanyl nucleotides in the absence of hormone. The coupling between the beta 1-adrenoceptor GS and C is efficient when compared with the coupling obtained in native membrane systems. GTP/GDP exchange at the alpha S subunit requires the presence of the beta gamma subunits. A mechanism for the inhibition of adenylate cyclase by the inhibitory GTP regulatory protein Gi is suggested.

TPK1, a Ca(2+)-regulated Arabidopsis vacuole two-pore K(+) channel is activated by 14-3-3 proteins.

The vacuole represents a pivotal plant organelle for management of ion homeostasis, storage of proteins and solutes, as well as deposition of cytotoxic compounds. Ion channels, pumps and carriers in the vacuolar membrane under control of cytosolic factors provide for ionic and metabolic homeostasis between this storage organelle and the cytoplasm. Here we show that AtTPK1 (KCO1), a vacuolar membrane localized K(+) channel of the TPK family, interacts with 14-3-3 proteins (general regulating factors, GRFs). Following in planta expression TPK1 and GRF6 co-localize at the vacuolar membrane. Co-localization of wild-type TPK1, but not the TPK1-S42A mutant, indicates that phosphorylation of the 14-3-3 binding motif of TPK1 represents a prerequisite for interaction. Pull-down assays and surface plasmon resonance measurements revealed GRF6 high-affinity interaction with TPK1. Following expression of TPK1 in yeast and isolation of vacuoles, patch-clamp studies identified TPK1 as a voltage-independent and Ca(2+)-activated K(+) channel. Addition of 14-3-3 proteins strongly increased the TPK1 activity in a dose-dependent manner. However, an inverse effect of GRF6 on the activity of the slow-activating vacuolar (SV) channel was observed in mesophyll vacuoles from Arabidopsis thaliana. Thus, TPK1 seems to provide for a Ca(2+)- and 14-3-3-sensitive mechanism capable of controlling cytoplasmic potassium homeostasis in plants.

Proteolysis-associated deglycosylation of beta 1-adrenergic receptor in turkey erythrocytes and membranes.

A protease that can be inhibited by glutathione, dithiothreitol, and o-phenanthroline but not by ethylenediaminetetraacetic acid converts the 50-kilodalton beta-adrenergic receptor in turkey erythrocyte membranes to a 40-kDa polypeptide which retains the specific ligand binding site. This conversion is attenuated in intact erythrocytes. The large 50-kDa peptide contains N-linked, complex carbohydrates and is retained on wheat germ agglutinin-Sepharose. The 40-kDa product of proteolysis does not bind to the wheat germ agglutinin and can thus be separated from the 50-kDa polypeptide by lectin chromatography. However, the large difference in molecular weights of the two receptor peptides cannot be accounted for solely by the different extent of glycosylation.

Multisite contacts involved in coupling of the beta-adrenergic receptor with the stimulatory guanine-nucleotide-binding regulatory protein. Structural and functional studies by beta-receptor-site-specific synthetic peptides.

Synthetic peptides, 12-22 amino acid residues long, comprising the presumed coupling sites of the beta-adrenergic receptor with the stimulatory guanine-nucleotide-binding regulatory protein (Gs), were examined for their ability to modulate Gs activation in turkey erythrocyte membranes. Three peptides corresponding to the second cytoplasmic loop, the N-terminal region of the third cytoplasmic loop, and the N-terminal region of the putative fourth cytoplasmic loop, compete synergistically with the hormone-stimulated receptor for Gs activation with median effector concentrations of 15-35 microM, or 3-4 microM for combinations of two peptides. One peptide, corresponding to the C-terminal region of the third cytoplasmic loop, carries the unique ability to activate the Gs-adenylate-cyclase complex independent of the signalling state of the receptor. These observations are consistent with a dynamic model of receptor-mediated G-protein activation in membranes, where domains composed of the second, third and fourth intracellular loop of the receptor bind to and are interactive with the G-protein heterotrimer, resulting in ligand-induced conformational changes of the receptor. In response to hormone binding, the extent or the number of sites involved in interaction with Gs may be readjusted using a fourth site. Modulation of coupling sites may elicit congruent conformational changes within the Gs heterotrimer, with qualitatively different effects on GTP/GDP exchange in the alpha subunit of Gs and downstream effector regulation. This model corroborates and expands a similar model suggested for activated rhodopsin-transducin interaction [König, B., Arendt, A., McDowell, J. H., Kahlert, M., Hargrave, P. A. & Hofmann, K. P. (1989) Proc. Natl Acad. Sci. USA 86, 6878-6882].

Specific enhancement of beta-adrenergic receptor kinase activity by defined G-protein beta and gamma subunits.

The beta and gamma subunits of heterotrimeric guanine nucleotide-binding proteins (G proteins) have recently been shown to play an active role in signal transduction. Among other effects they enable translocation of the beta-adrenergic receptor kinase (beta ARK) from the cytosol to the plasma membrane and thus permit phosphorylation and ultimately desensitization of beta-adrenergic receptors and other G-protein-coupled receptors. To investigate the specificity of this effect, we have purified various combinations of recombinant beta and gamma subunits expressed in Sf9 cells and measured their effects on beta ARK-catalyzed phosphorylation of beta 2-adrenergic receptors and of rhodopsin. The combinations tested were beta 1 gamma 2, beta 1 gamma 3, beta 2 gamma 2, beta 2 gamma 3, and transducin beta gamma (beta 1 gamma 1). There were clear differences in enhancement of rhodopsin phosphorylation, with an order of efficacy beta 2 gamma 2 > beta 1 gamma 2 >> beta 2 gamma 3 approximately beta 1 gamma 3 approximately beta 1 gamma 1. The first two combinations had larger effects than a mixed beta gamma preparation from bovine brain. In enhancing phosphorylation of beta 2-adrenergic receptors, beta 1 gamma 2 was more efficient and potent than all other combinations. These data suggest a twofold specificity of beta gamma complexes in enhancing beta ARK-catalyzed receptor phosphorylation: the gamma subunits may be important in interacting with beta ARK, with gamma 2 being more potent than gamma 3, whereas the beta subunits may determine coupling to the receptors, with beta 2 being more effective than beta 1 for rhodopsin and beta 1 being more effective than beta 2 for beta 2-adrenergic receptors.

Photoaffinity labeling of the beta-adrenergic receptor with azide derivatives of iodoccyanopindolol.

Two photosensitive iodocyanopindolol derivatives, 1-(4-azidobenzimidyl)-3,3-dimethyl-6-hydroxy-7-(2-cyano-3-iodoindol-4-yloxy)-1,4-diazaheptane (ICYP-azide-1) and 1-(4-azidobenzoyl)-3,3-dimethyl-6-hydroxy-7-(2-cyano-3-iodoindol-4-yloxy)-1,4-diazaheptane (ICYP-azide-2) have been prepared. [125I]ICYP-azide-1 and -2 (specific radioactivity up to 2.2 Ci/mumol) bind specifically and with very high affinity (KD = 40-45 pM) to beta-adrenergic receptors of turkey erythrocyte membranes. When [125I]ICYP-azide-1 or -2 were incubated with membranes and UV-irradiated, two polypeptides (Mr = 40,000 and 50,000) were specifically photolabeled as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These polypeptides may represent subunits of the beta-adrenergic receptor. The yield of specific covalent label incorporation into both polypeptides was up to 17.2% with [125I]ICYP-azide-2 when expressed as fraction of total beta-receptor binding sites. Since the Mr = 40,000 polypeptide was labeled predominantly and since covalent incorporation had the same concentration dependence as reversible specific binding, this polypeptide could contain a beta-adrenergic ligand binding site. Due to the low working concentration (10-100 pM) of [125I]ICYP-azide-1 and -2, nonspecific labeling of membrane proteins was extremely low. The new photoaffinity labels should therefore become valuable tools for probing beta-receptor structure.

Functional and structural characterization of the two beta 1-adrenoceptor forms in turkey erythrocytes with molecular masses of 50 and 40 kilodaltons.

We have previously described a specific protease in turkey erythrocytes that converts the larger 50-kDa (P50) form of the beta 1-adrenoceptor to a smaller 40-kDa (P40) form [Jürss, R., Hekman, M., & Helmreich, E. J. M. (1985) Biochemistry 24, 3349-3354]. Further functional and structural characterization studies of the two forms are reported here. When purified P50 and P40 receptors were compared with respect to their relative capabilities to couple in lipid vesicles with pure stimulatory G-proteins (Gs-proteins) prepared from turkey erythrocytes or rabbit liver, a faster and larger activation of Gs-proteins was observed in response to l-isoproterenol and guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) with P40 than with P50 receptor. The kon values for P40 were 0.47 min-1 in the case of liver Gs and 0.22 min-1 in the case of erythrocyte Gs, whereas the corresponding values for P50 were 0.34 min-1 and 0.12 min-1, respectively. The binding properties of P50 and P40 forms of the receptor were not different, and desensitization of turkey erythrocytes on exposure to l-isoproterenol did not activate the protease. We furthermore ascertained that only the larger form with a molecular mass of 50 kDa carries the N-linked carbohydrates, which are removed on proteolytic conversion to the 40-kDa form and have either a triantennary or a tetraantennary nonfucosylated complex-type structure containing terminal sialyl residues.

Role of glycosylation for beta 2-adrenoceptor function in A431 cells.

A431 cells incubated with tunicamycin (0.15 micrograms/ml) for 40 h under conditions where incorporation of [3H] leucine into protein was inhibited less than 10% expressed mainly a beta-receptor species of about Mr 40,000 which was ascribed to the nonglycosylated form of the beta-receptor of about Mr 75,000 found in normal A431 cells by photoaffinity labeling. However, the tunicamycin-treated cells expressed the same number of specific beta 2-receptor-binding sites as untreated cells. Moreover, the aglycoreceptors had the same ligand binding properties as beta-adrenoceptors from control cells; but, functional tests of the receptor from tunicamycin-treated cells in reconstituted lipid vesicles showed that receptors from tunicamycin-treated cells had lost coupling efficiency. The coupling defect was at the receptor level since control experiments indicated that the other components of the signal transmission chain from beta-adrenoceptor to adenylate cyclase, the stimulatory regulatory GTP-binding protein of adenylate cyclase and adenylate cyclase, were fully functional. Homologous desensitization in tunicamycin-treated cells was characterized by export from the cell surface and sequestration of about the same number of beta-adrenoceptors as in normal desensitized cells but without further reduction of hormonally stimulated adenylate cyclase below the low level already attained in nondesensitized tunicamycin-treated cells. This was explained by assuming that the receptors removed in the course of homologous desensitization from the surface of tunicamycin-treated cells were already nonfunctional. Thus, beta-adrenergic desensitization in tunicamycin-treated cells is characterized by the functional disengagement of receptor removal and loss of adenylate cyclase activity.

Binding of purified recombinant beta-arrestin to guanine-nucleotide-binding-protein-coupled receptors.

beta-arrestin is a cytosolic protein thought to be responsible for uncoupling agonist-activated beta 2-adrenergic receptors from their guanine-nucleotide-binding proteins (G-protein) subsequent to receptor phosphorylation by the beta-adrenergic receptor kinase (beta ARK). In order to investigate this interaction, we generated a recombinant baculovirus for the expression of beta-arrestin in Sf9 insect cells. Apparently homogeneous beta-arrestin preparations were obtained in a one-step purification on heparin-Sepharose. Purified beta-arrestin bound to rhodopsin in a phosphorylation-dependent plus light-dependent manner. Binding to beta 2-adrenergic receptors was investigated using purified receptors reconstituted into lipid vesicles. The accessibility of the reconstituted receptors was determined using the agonist isoproterenol for the ligand-binding site and an antibody binding to an attached myc tag for the C-terminus, the site of receptor phosphorylation. On the basis of these data, the binding of purified beta-arrestin to beta ARK-phosphorylated beta 2-adrenergic receptors was found to occur with a KD of 1.8 nM and with a maximum of 1 beta-arrestin/receptor. beta-arrestin also bound to receptors which had been completely dephosphorylated with acid phosphatase, but the affinity was approximately 30-fold lower. In contrast to regulation by phosphorylation, binding of agonists or antagonists to the receptors had negligible effects on beta-arrestin binding. Finally, beta-arrestin and beta ARK were shown to be capable of producing synergistic inhibition of beta 2-adrenergic-receptor-stimulated adenylyl cyclase activity of cell membranes. These data show that high-affinity stoichiometric binding of beta-arrestin to beta 2-adrenergic receptors occurs in a beta ARK-dependent manner and is sufficient to impair adenylyl cyclase stimulation by the receptors.

Lateral motion of beta receptors in membranes of cultured liver cells.

We have studied the lateral mobility and distribution of beta receptors on Chang human liver cells by fluorescence photobleaching recovery and video intensification microscopy. The beta receptors were labeled with the fluorescent antagonist 7-(2-allylphenoxy)-2,2-dimethyl-6-hydroxy-1-(4-nitrobenzo-2-oxa-1,3-diazolyl)-1 ,4-diazaheptane (Alp-NBD). Sixty to 75% of the staining was specific (displaceable by unlabeled antagonists). Most of the antagonist-occupied beta receptors were immobile, because only 15-25% of their fluorescence recovered on the experimental time scale at 23 degrees C. This immobility correlates with the clustered distribution of Alp-NBD--beta-receptor complexes at 4 degrees C and 37 degrees C. The beta receptors appear to be aggregated prior to antagonist binding, because visible patches were observed immediately after labeling for 30 sec at 4 degrees C. Preincubation at 37 degrees C with (--)-isoproterenol, a beta agonist, prior to Alp-NBD labeling induced a time-dependent release of the beta receptors to a more homogeneous distribution and increased the mobile fraction to 70-80% (lateral diffusion coefficient = 1.4 X 10(-9) cm2/sec at 23 degrees C). This is not due to an effect on membrane fluidity, because the diffusion coefficient of a lipid probe was not altered. The time course of agonist-induced beta-receptor mobilization correlates with receptor loss and adenylate cyclase desensitization but is much slower than adenylate cyclase activation. This indicates that adenylate cyclase activation by beta receptors does not require macroscopic lateral mobility of the majority of the beta receptors.

Regulation of signal transfer from beta 1-adrenoceptor to adenylate cyclase by beta gamma subunits in a reconstituted system.

The beta gamma subunits of guanine nucleotide binding proteins from bovine brain and bovine rod outer segments have different structural and immunochemical properties. In spite of these structural differences, beta gamma subunits from these sources have been found to be fully interchangeable in terms of their interaction with alpha subunits of pertussis-toxin-sensitive G proteins. In contrast, however, there are striking differences between these beta gamma subunits with regard to their ability to deactivate fluoride-stimulated Gs. These profound differences were also observed when the interaction of the purified components of the adenylate cyclase system was studied after reconstitution into phospholipid vesicles. Addition of beta gamma purified from bovine brain to vesicles containing beta-receptor and Gs results in a biphasic effect on receptor-stimulated GTPase activity, whereas addition of transducin beta gamma was virtually without any effect. Likewise, beta gamma from bovine brain, but not transducin beta gamma, affected adenylate cyclase activity of a reconstituted system consisting of three purified components (R, Gs, C). Thus, the alpha subunit of Gs, but not the alpha subunits of pertussis-toxin-sensitive G proteins discriminate between structurally different beta gamma subunits.