Genetics of a-agglutunin function in Saccharomyces cerevisiae

The Saccharomyces cerevisiae cell adhesion protein a-agglutinin is composed of an anchorage subunit (Aga1p) and an adhesion subunit (Aga2p). Although functional a-agglutinin is expressed only by a cells, previous results indicated that AGA1 RNA is expressed in both a and α cells after pheromone induction. Expression of the Aga2p adhesion subunit in a cells allowed a-agglutinability, indicating that a cells express the a-agglutinin anchorage subunit, although no role for Aga1p in α cells has been identified. Most of the a-specific agglutination-defective mutants isolated previously were defective in AGA1; a single mutant (La199) was a candidate for an aga2 mutant. Expression of AGA2 under PGK control allowed secretion of active Aga2p from control strains but did not complement the La199 agglutination defect or allow secretion of Aga2p from La 199, suggesting that the La199 mutation might identify a new gene required for a-agglutinin function. However, the La199 agglutination defect showed tight linkage to aga2::URA3 and did not complement aga2::URA3 in a/a diploids. The aga2 gene cloned from La199 was nonfunctional and contained an ochre mutation. The inability of pPGK-AGA2 to express functional Aga2p in La199 was shown to result from an additional mutation(s) that reduces expression of plasmid-borne genes. AGA2 was mapped to the left arm of chromosome VII approximately 28 cM from the centromere.     

Brefeldin a down-regulates the transferrin receptor in K562 cells

The fungal metabolite brefeldin A (BFA) induces profound alterations in the morphology of intracellular organelles. Although BFA promotes the formation of extensive tubular endosomal domains, our understanding of the effects of the antibiotic on vesicle traffic events associated with endocytosis is limited. Thus, alterations in the transferrin (Tf) receptor's endocytic/recycling pathway upon treatment of human erythroleukemia K562 cells with BFA were studied as a pharmacological response. Treatment of K562 cells with BFA caused a down-regulation in the number of cell surface Tf receptors. This effect is highly reminiscent of the well-known action of phorbol 12-myristate 13-acetate (PMA) on Tf receptor traffic in K562 cells. However, our results demonstrate that these two agents down-regulate the Tf receptor via different mechanisms. The effects of BFA and PMA were additive when K562 cells were incubated with both together. Using the In/Sur method, the endocytic rate constant for Tf internalization was determined and PMA was found to greatly enhance k_e, from 0.28 min^–1 to 0.43 min^–1, while BFA had little effect (K_e=0.20 min^–1). In contrast, BFA-treatment alters the exocytic rate constant for return of internalized receptors to the cell surface, with the largest effect exerted on a slow-release, monensin-sensitive, compartment. The sum of the endocytic and exocytic kinetic data support a model in which BFA and PMA down-regulate the Tf receptor in K562 cells by mechanistically distinct actions, with BFA targeting exocytic monensin-sensitive intracellular compartments and PMA acting to exert a profound influence on elements of receptor internalization.Abbreviations BFA brefeldin A - ARF ADP-ribosylation factor - HRP horseradish peroxidase - Tf transferrin - PMA phorbol 12-myristate 13-acetate - DMSO dimethyl sulfoxide - PBS phosphate-buffered saline - HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - BSA bovine serum albumin - FITC-Tf fluorescein isothiocyanate-labelled transferrin     

Aerodynamic corrections for the flight of birds and bats in wind tunnels

Few wind tunnel studies of animal flight have controlled or corrected for distortions to behaviour, physiology or flight aerodynamics representing the difference between flight in the tunnel and flight in free air. Aerodynamic correction factors are derived based on lifting-line theory and the method of images for an animal flying freely within closed- and open-section wind tunnels; the method is very similar to that used to model flight in ground effect, and as in ground effect the corrections to induced drag may be substantial. These correction factors are used to estimate bound wing circulation, drag and mechanical power for comparison with free flight, and to derive testable predictions of optimum flight strategies for an animal in a tunnel. In an open-section tunnel, mechanical power is increased compared to free flight, and the animal should fly at the tunnel centre. In a closed tunnel mechanical power is usually reduced, and substantial savings are available, particularly at low speeds, if the animal flies close to the tunnel roof. Anecdotal observations confirm that birds and bats adopt this strategy. The mechanical power-speed curve in a closed tunnel is flatter than the curve for free flight, and this may explain the flat metabolic power-speed curves for birds and bats obtained in some measurements.     

The biodegradation of piperazine and structurally-related linear and cyclic amines

The biodegradability of a range of linear and cyclic amines was assessed. All proved to be biodegradable but there were interesting differences in their susceptibility. The least degradable was piperazine although piperazine-degrading microorganisms were of widespread occurrence in samples of water and activated sludge and, to a lesser extent, soils. Piperazine degraders are only present in very small numbers — on averageca. 0.8/ml of river water. Of six isolates capable of using piperazine as a sole source of carbon, nitrogen and energy in pure culture five were identified asMycobacterium spp. and one asArthrobacter sp., all strains were capable only of slow growth (mean generation time ofca. 30 to 40 hours) on this substrate. Piperidine, pyrrolidine, ethanolamine and diethanolamine were all readily biodegradable. The relationship between structure and degradability of amines is discussed as are the possible reasons for the relative recalcitrance of piperazine.     

Aberrant protein phosphorylation at tyrosine is responsible for the growth-inhibitory action of pp60v-src expressed in the yeast Saccharomyces cerevisiae.

Expression of pp60v-src, the transforming protein of Rous sarcoma virus, arrests the growth of the yeast Saccharomyces cerevisiae. To determine the basis of this growth arrest, yeast strains were constructed that expressed either wild-type v-src or various mutant v-src genes under the control of the galactose-inducible, glucose repressible GAL1 promoter. When shifted to galactose medium, cells expressing wild-type v-src ceased growth immediately and lost viability, whereas cells expressing a catalytically inactive mutant (K295M) continued to grow normally, indicating that the kinase activity of pp60v-src is required for its growth inhibitory effect. Mutants of v-src altered in the SH2/SH3 domain (XD4, XD6, SPX1, and SHX13) and a mutant lacking a functional N-terminal myristoylation signal (MM4) caused only a partial inhibition of growth, indicating that complete growth inhibition requires either targeting of the active kinase or binding of the kinase to phosphorylated substrates, or both. Cells arrested by v-src expression displayed aberrant microtubule structures, alterations in DNA content and elevated p34CDC28 kinase activity. Immunoblotting with antiphosphotyrosine antibody showed that many yeast proteins, including the p34CDC28 kinase, became phosphorylated at tyrosine in cells expressing v-src. Both the growth inhibition and the tyrosine-specific protein phosphorylation observed following v-src expression were reversed by co-expression of a mammalian phosphotyrosine-specific phosphoprotein phosphatase (PTP1B). However a v-src mutant with a small insertion in the catalytic domain (SRX5) had the same lethal effect as wild-type v-src, yet induced only very low levels of protein-tyrosine phosphorylation. These results indicate that inappropriate phosphorylation at tyrosine is the primary cause of the lethal effect of pp60v-src expression but suggest that only a limited subset of the phosphorylated proteins are involved in this effect.     

DNA-binding domain of bovine papillomavirus type 1 E1 helicase: structural and functional aspects.

The E1 protein of bovine papillomavirus type 1 is a multifunctional enzyme required for papillomaviral DNA replication. It assists in the initiation of replication both as a site-specific DNA-binding protein and as a DNA helicase. Previous work has indicated that at limiting E1 concentrations, the E2 protein is required for efficient E1 binding to the replication origin. In this study, we have defined the domain of the E1 protein required for site-specific DNA binding. Experiments with a series of truncated proteins have shown that the first amino-terminal 299 amino acids contain the DNA-binding domain; however, the coterminal M protein, which is homologous to E1 for the first 129 amino acids, does not bind origin DNA. A series of small internal deletions and substitution mutations in the DNA-binding domain of E1 show that specific basic residues in this region of the protein, which are conserved in all E1 proteins of the papillomavirus family, likely play a direct role in binding DNA and that a flanking conserved hydrophobic subdomain is also important for DNA binding. A region of E1 that interacts with E2 for cooperative DNA binding is also retained in carboxy-terminal truncated proteins, and we show that the ability of full-length E1 to complex with E2 is sensitive to cold. The E1 substitution mutant proteins were expressed from mammalian expression vectors to ascertain whether site-specific DNA binding by E1 is required for transient DNA replication in the cell. These E1 proteins display a range of mutant phenotypes, consistent with the suggestion that site-specific binding by E1 is important. Interestingly, one E1 mutant which is defective for origin binding but can be rescued for such activity by E2 supports significant replication in the cell.     

Receptor-Mediated Stimulation and Inhibition of Nerve Growth Factor Secretion by Vascular Smooth Muscle

Nerve growth factor (NGF) is a potent neurotrophin signaling protein, the best-known member of a family of similar neurotrophins. Specific neuronal populations depend upon the neurotrophins for normal function and disturbances in NGF and neurotrophin supply have been implicated in neurodegenerative disease, diabetes, and hypertension. This report details experiments in which the hourly pattern of NGF secretion by cultured vascular smooth muscle cells is examined. Vascular smooth muscle cells are major innervation targets of the neuronal population first discovered to be NGF-dependent: the sympathetic principal neurons. The results show that arginine vasopressin (AVP), angiotensin II (AngII), and α-adrenergic receptor activation, all contractile stimuli, elevate NGF secretion. However, AVP dependably does so alone while AngII requires coactivation of adenosine receptors. Adenosine alone inhibits secretion and the α-adrenergic increase in NGF output can be antagonized by activation of β-adrenergic receptors. A change to fresh culture medium is also a potent stimulus to increased NGF output.