Genetic and biochemical analysis of the adenylyl cyclase-associated protein, cap, in Schizosaccharomyces pombe

We have identified, cloned, and studied a gene, cap, encoding a protein that is associated with adenylyl cyclase in the fission yeast Schizosaccharomyces pombe. This protein shares significant sequence homology with the adenylyl cyclase-associated CAP protein in the yeast Saccharomyces cerevisiae. CAP is a bifunctional protein; the N-terminal domain appears to be involved in cellular responsiveness to RAS, whereas loss of the C-terminal portion is associated with morphological and nutritional defects. S. pombe cap can suppress phenotypes associated with deletion of the C-terminal CAP domain in S. cerevisiae but does not suppress phenotypes associated with deletion of the N-terminal domain. Analysis of cap disruptants also mapped the function of cap to two domains. The functional loss of the C-terminal region of S. pombe cap results in abnormal cellular morphology, slow growth, and failure to grow at 37-degrees-C. Increases in mating and sporulation were observed when the entire gene was disrupted. Overproduction of both cap and adenylyl cyclase results in highly elongated large cells that are sterile and have measurably higher levels of adenylyl cyclase activity. Our results indicate that cap is required for the proper function of S. pombe adenylyl cyclase but that the C-terminal domain of cap has other functions that are shared with the C-terminal domain of S. cerevisiae CAP

Localization of thioredoxin in the rat brain and functional implications

The immunoreactivity for thioredoxin, which catalyzes protein disulfide reductions, has previously been shown to exist in nerve cells and their axons. Here we demonstrate the localization of thioredoxin mRNA as revealed by in situ hybridization in the rat brain. The gene is expressed in nerve cells of a variety of brain regions, for example, the cerebral cortex, the piriform cortex, the medial preoptic area, the CA3/CA4 region of the hippocampal formation, the dentate gyrus, the paraventricular nucleus of the hypothalamus, the arcuate nucleus, the substantia nigra pars compacta, the locus coeruleus, the ependyma of the 4th ventricle, and the epithelial cells of the choroid plexus. This distribution implicates an important function in nerve cell metabolism, especially in regions with high energy demands and indicates a role of the choroid plexus in nerve cell protection from environmental influences. It was found that after mechanical injury induced by partial unilateral hemitransection the thioredoxin mRNA expression is upregulated in the lesioned area and spreads to the cortical hemispheres at the lesioned level. This induction suggests a function of thioredoxin in the regeneration machinery of the brain following mechanical injury and oxidative stress

Negative regulation of syntaxin4/SNAP-23/VAMP2-mediated membrane fusion by Munc18c <i>In Vitro</i>

Background: Translocation of the facilitative glucose transporter GLUT4 from an intracellular store to the plasma membrane is responsible for the increased rate of glucose transport into fat and muscle cells in response to insulin. This represents a specialised form of regulated membrane trafficking. Intracellular membrane traffic is subject to multiple levels of regulation by conserved families of proteins in all eukaryotic cells. Notably, all intracellular fusion events require SNARE proteins and Sec1p/Munc18 family members. Fusion of GLUT4-containing vesicles with the plasma membrane of insulin-sensitive cells involves the SM protein Munc18c, and is regulated by the formation of syntaxin 4/SNAP23/VAMP2 SNARE complexes. Methodology/Principal Findings Here we have used biochemical approaches to characterise the interaction(s) of Munc18c with its cognate SNARE proteins and to examine the role of Munc18c in regulating liposome fusion catalysed by syntaxin 4/SNAP23/VAMP2 SNARE complex formation. We demonstrate that Munc18c makes contacts with both t- and v-SNARE proteins of this complex, and directly inhibits bilayer fusion mediated by the syntaxin 4/SNAP23/VAMP2 SNARE complex. Conclusion/Significance Our reductionist approach has enabled us to ascertain a direct inhibitory role for Munc18c in regulating membrane fusion mediated by syntaxin 4/SNAP23/VAMP2 SNARE complex formation. It is important to note that two different SM proteins have recently been shown to stimulate liposome fusion mediated by their cognate SNARE complexes. Given the structural similarities between SM proteins, it seems unlikely that different members of this family perform opposing regulatory functions. Hence, our findings indicate that Munc18c requires a further level of regulation in order to stimulate SNARE-mediated membrane fusion

Automation of Pivot Sprinkler Irrigation Systems to More Efficiently Utilize Rainfall and Irrigation Water

A study was conducted to develop automated pivot sprinkler irrigation systems and determine if such systems use less water and energy than manually operated systems. The study was conducted near Earth, Texas, using irrigation systems located on producers farms. Sensors with transmitters and receivers were constructed and tested so that the irrigation systems can be controlled by wind, soil water tension, and rainfall. The sensors can be used separately or in combination to control the irrigation systems. For several reasons it was not possible to determine if automated systems use less water and energy than manually operated systems. The major reason was the low capacity of the wells (114 to 204 m3/hr) supplying the irrigation systems. To meet crop water requirements and losses due to evaporation and runoff, the well capacity should be at least 284 m3/hr. Since the wells could not supply adequate water, soil water tension was out of the tensiometer range for the last 60 days of the growing season. Considerable variation in soil water tension and content was noted between irrigation systems and within quadrants of each irrigation system. Systems planted to cotton would probably be easier to automate than those planted in corn because of the lower water requirements of cotton. The wind and rainfall controls have more promise to aid in increasing water use efficiency than controls activated by soil water sensors. Wind controls could be used during preirrigation when more time is available to apply water and rainfall controls could be an aid to producers with remotely located irrigation systems

Hearing shapes of drums - mathematical and physical aspects of isospectrality

In a celebrated paper '"Can one hear the shape of a drum?"' M. Kac [Amer. Math. Monthly 73, 1 (1966)] asked his famous question about the existence of nonisometric billiards having the same spectrum of the Laplacian. This question was eventually answered positively in 1992 by the construction of noncongruent planar isospectral pairs. This review highlights mathematical and physical aspects of isospectrality.Comment: 42 pages, 60 figure

Analysis of SEC9 Suppression Reveals a Relationship of SNARE Function to Cell Physiology

BACKGROUND:Growth and division of Saccharomyces cerevisiae is dependent on the action of SNARE proteins that are required for membrane fusion. SNAREs are regulated, through a poorly understood mechanism, to ensure membrane fusion at the correct time and place within a cell. Although fusion of secretory vesicles with the plasma membrane is important for yeast cell growth, the relationship between exocytic SNAREs and cell physiology has not been established. METHODOLOGY/PRINCIPAL FINDINGS:Using genetic analysis, we identified several influences on the function of exocytic SNAREs. Genetic disruption of the V-ATPase, but not vacuolar proteolysis, can suppress two different temperature-sensitive mutations in SEC9. Suppression is unlikely due to increased SNARE complex formation because increasing SNARE complex formation, through overexpression of SRO7, does not result in suppression. We also observed suppression of sec9 mutations by growth on alkaline media or on a non-fermentable carbon source, conditions associated with a reduced growth rate of wild-type cells and decreased SNARE complex formation. CONCLUSIONS/SIGNIFICANCE:Three main conclusions arise from our results. First, there is a genetic interaction between SEC9 and the V-ATPase, although it is unlikely that this interaction has functional significance with respect to membrane fusion or SNAREs. Second, Sro7p acts to promote SNARE complex formation. Finally, Sec9p function and SNARE complex formation are tightly coupled to the physiological state of the cell

Shell evolution of N = 40 isotones towards 60Ca: First spectroscopy of 62Ti

Excited states in the N=40 isotone 62Ti were populated via the 63V(p,2p)62Ti reaction at ∼200 MeV/nucleon at the Radioactive Isotope Beam Factory and studied using γ-ray spectroscopy. The energies of the 21+→0gs+ and 41+→21+ transitions, observed here for the first time, indicate a deformed 62Ti ground state. These energies are increased compared to the neighboring 64Cr and 66Fe isotones, suggesting a small decrease of quadrupole collectivity. The present measurement is well reproduced by large-scale shell-model calculations based on effective interactions, while ab initio and beyond mean-field calculations do not yet reproduce our findings. The shell-model calculations for 62Ti show a dominant configuration with four neutrons excited across the N=40 gap. Likewise, they indicate that the N=40 island of inversion extends down to Z=20, disfavoring a possible doubly magic character of the elusive 60Ca