In addition to the SH3 binding region, multiple regions within the N-terminal noncatalytic portion of the cAMP-specific phosphodiesterase, PDE4A5, contribute to its intracellular targeting

The long cyclic AMP (cAMP)-specific phosphodiesterase isoform, PDE4A5 (PDE4A subfamily isoform variant 5), when transiently expressed in COS-7 cells, was shown in subcellular fractionation studies to be associated with both membrane and cytosol fractions, with immunofluorescence analyses identifying PDE4A5 as associated both with ruffles at the cell margin and also at a distinct perinuclear localisation. Deletion of the first nine amino acids of PDE4A5 (1) ablated its ability to interact with the SH3 domain of the tyrosyl kinase, LYN; (2) reduced, but did not ablate, membrane association; and (3) disrupted the focus of PDE4A5 localisation within ruffles at the cell margin. This deleted region contained a Class I SH3 binding motif of similar sequence to those identified by screening a phage display library with the LYN-SH3 domain. Truncation to remove the PDE4A5 isoform-specific N-terminal region caused a further reduction in membrane association and ablated localisation at the cell margin. Progressive truncation to delete the PDE4A long isoform common region and then the long isoform-specific UCR1 did not cause any further change in membrane association or intracellular distribution. However, deletion up to the super-short form splice junction generated an entirely soluble 'core' PDE4A species. We propose that multiple sites in the N-terminal noncatalytic portion of PDE4A5 have the potential to associate with intracellular structures and thus define its intracellular localisation. At least two such sites lie within the PDE4A5 isoform-specific N-terminal region and these appear to be primarily responsible for targeting PDE4A5 to, and organising it within, the cell margin; one is an SH3 binding motif able to interact with LYN kinase and the other lies within the C-terminal portion of the PDE4A5 unique region. A third membrane association region is located within the N-terminal portion of UCR2 and appears to be primarily responsible for targeting to the perinuclear region. Progressive N-terminal truncation, to delete defined regions of PDE4A5, identified activity changes occurring upon deletion of the SH3 binding site region and then upon deletion of the membrane association site region located within UCR2. This suggests that certain of these anchor sites may not only determine intracellular targeting but may also transduce regulatory effects on PDE4A5 activity.     

Study of the interaction of Saccharomyces cerevisiae with glucose by particle microelectrophoresis

Saccharomyces cerevisiae NCYC 239 in the presence of glucose at temperatures under 303 K shows a time-dependent lowering of electrophoreric mobility v. At temperatures above 303 K, this time-dependent change in v is in the direction of increased mobilities. Cells suspended in buffer indicate a surface pKa of less than 4, whereas for cells suspended in buffered glucose it is impossible to derive a surface pKa. A kinetic study of the interaction of S. cerevisiae with glucose as a function of temperature allows calculation of an activation energy of 140 kJ X mol-1 for the combined processes of (i) uptake of glucose onto the cell wall, (ii) transfer through the cell wall and membrane, and (iii) the establishment of a steady glucose flux through the wall and membrane.     

Intraluminal transport of iron from stomach to small-intestinal mucosa

Inorganic iron rarely exceeds 10⁻⁴ molar concentration in the stomach after a meal. Natural sugars, ascorbic acid, citric acid, and amino-acids form iron complexes, and if they are present in the meal complexing occurs when the gastric contents are neutralized. In their absence an iron complex is formed with gastric mucopolysaccharide, which acts as a carrier, stable at neutral pH. Iron can be detached from this carrier at neutral pH by some low molecular weight substances, of which citric acid, ascorbic acid, and cysteine are particularly effective at low concentrations. Under normal circumstances most of the iron released from food in the stomach becomes bound to the mucopolysaccharide carrier.     

Transcriptional profiling of Zea mays roots reveals roles for jasmonic acid and terpenoids in resistance against Phytophthora cinnamomi

Phytophthora cinnamomi is a soil-borne plant pathogen that has caused widespread damage to vulnerable native ecosystems and agriculture systems across the world and shows no sign of abating. Management of the pathogen in the natural environment is difficult and the options are limited. In order to discover more about how resistant plants are able to defend themselves against this generalist pathogen, a microarray study of plant gene expression following root inoculation with P. cinnamomi was undertaken. Zea mays was used as a resistant model plant, and microarray analysis was conducted using the Affymetrix GeneChip Maize Genome Array on root samples collected at 6- and 24-h post-inoculation. Over 300 genes were differentially expressed in inoculated roots compared with controls across the two time points. Following Gene Ontology enrichment analysis and REVIGO visualisation of the up-regulated genes, many were implicated in plant defence responses to biotic stress. Genes that were up-regulated included those involved in phytoalexin biosynthesis and jasmonic acid/ethylene biosynthesis and other defence-related genes including those encoding glutathione S-transferases and serine-protease inhibitors. Of particular interest was the identification of the two most highly up-regulated genes, terpene synthase11 (Tps11) and kaurene synthase2 (An2), which are both involved in production of terpenoid phytoalexins. This is the first study that has investigated gene expression at a global level in roots in response to P. cinnamomi in a model plant species and provides valuable insights into the mechanisms involved in defence.     

Distribution of uterine histological changes in aged captive cheetahs (Acinonyx jubatus)

The histological effect on the felid uterus of sterilization, via ovariectomy or salpingectomy, is currently unknown. To investigate the association of ovariectomy or salpingectomy with uterine health, it is first necessary to establish if changes are distributed evenly throughout the uterus. Both laparoscopic ovariectomy and salpingectomy with concurrent sampling of the tip of the uterine horn are possible in the cheetah. Currently accepted practice for histopathological screening of the uterus utilizes four biopsy samples. It is not known whether this method accurately reflects the status of the entire uterus. In this study we histologically examined the uteri of six older cheetahs (one 7-year-old and five 10–10.5-year-old animals) via 21 tissue samples (three samples from seven different anatomical regions) per cheetah to determine overall uterine health. Although no defined lesions were detected, mild endometrial gland dilation, assumed to be of no functional consequence, was observed in multiple samples. The odds of observing this dilation was lowest in the uterine body and progressively increased in a cranial direction, being significantly higher at the tip of the uterine horns (OR = 11.5; 95% CI, 2.0-65.1; p = 0.006). This supported the reliability of sampling the tip of the uterine horn to screen for endometrial gland dilation.     

Enzymatic Synthesis of s-Substituted l-Cysteines with Tryptophan Synthase of Escherichia coli

The α₂β₂ complex of tryptophan synthase from Escherichia coli catalyzes β-replacement reactions of l-serine and its derivatives (e.g., β-chloro-l-alanine and O-methyl-Dl-serine) with various alkanethiols. The products from thiobenzyl alcohol and ethanethiol were isolated to demonstrate the enzymatic synthesis of the corresponding S-substituted l-cysteines. Reactivities of various S-substituent donors were examined, and thiols such as thiobenzyl alcohol, 1-propanethiol and 1-butanethiol were found to be much more efficient substituent donors than the physiological substrate, indole. In addition, tryptophan synthase catalyzes β-replacement reactions of l-threonine with thiols to form the corresponding S-substituted β-methylcysteines, which are also produced by β-addition reactions of l-vinylglycine with thiols. These enzymatic reactions facilitate the synthesis of various sulfur-containing amino acids.     

PyMINEr Finds Gene and Autocrine-Paracrine Networks from Human Islet scRNA-Seq

1. Download high-res image (260KB)
2. Download full-size image