Sir2-dependent activation of acetyl-CoA synthetase by deacetylation of active lysine

Acetyl-coenzyme A (CoA) synthetase (Acs) is an enzyme central to metabolism in prokaryotes and eukaryotes. Acs synthesizes acetyl CoA from acetate, adenosine triphosphate, and CoA through an acetyl-adenosine monophosphate (AMP) intermediate. Immunoblotting and mass spectrometry analysis showed that Salmonella enterica Acs enzyme activity is posttranslationally regulated by acetylation of lysine-609. Acetylation blocks synthesis of the adenylate intermediate but does not affect the thioester-forming activity of the enzyme. Activation of the acetylated enzyme requires the nicotinamide adenine dinucleotide-dependent protein deacetylase activity of the CobB Sir2 protein from S. enterica. We propose that acetylation modulates the activity of all the AMP-forming family of enzymes, including nonribosomal peptide synthetases, luciferase, and aryl- and acyl-CoA synthetases. These findings extend our knowledge of the roles of Sir2 proteins in gene silencing, chromosome stability, and cell aging and imply that lysine acetylation is a common regulatory mechanism in eukaryotes and prokaryotes.     

Alteration of stimulus-specific guard cell calcium oscillations and stomatal closing in Arabidopsis det3 mutant

Cytosolic calcium oscillations control signaling in animal cells, whereas in plants their importance remains largely unknown. In wild-type Arabidopsis guard cells abscisic acid, oxidative stress, cold, and external calcium elicited cytosolic calcium oscillations of differing amplitudes and frequencies and induced stomatal closure. In guard cells of the V-ATPase mutant det3, external calcium and oxidative stress elicited prolonged calcium increases, which did not oscillate, and stomatal closure was abolished. Conversely, cold and abscisic acid elicited calcium oscillations in det3, and stomatal closure occurred normally. Moreover, in det3 guard cells, experimentally imposing external calcium-induced oscillations rescued stomatal closure. These data provide genetic evidence that stimulus-specific calcium oscillations are necessary for stomatal closure.     

Evaluation of the induction of vasoactive mediators from equine digital vein endothelial cells by endotoxin

OBJECTIVE: To determine the effect of endotoxin (lipopolysaccharide [LPS]) on vasoactive mediator production by cultured equine digital vein endothelial cells (EDVECs). SAMPLE POPULATION: EDVECs obtained from forelimb digital veins of 7 healthy adult horses. PROCEDURES: EDVECs were incubated with or without LPS (1 microg/mL) for 0, 2, 4, 6, 22, and 24 hours. The EDVECs were incubated for 18 hours with LPS (10 pg/mL to 1 microg/mL) with or without ibuprofen, cycloheximide, or L-nitroarginine methyl ester. Medium concentrations of prostacyclin, cyclic guanosine monophosphate, endothelin-1, and thromboxane A(2) were determined. Changes in inducible nitric oxide synthase and cyclooxygenase-2 expression were determined. RESULTS: LPS stimulated mean 4.2- and 14.1-fold increases in EDVEC prostacyclin and cyclic guanosine monophosphate production, respectively, after 22 hours. These effects were LPS concentration-dependent (LPS concentrations that induced a response halfway between the maximum response and baseline of 1.50 and 1.22 ng/mL, respectively). The LPS-induced cyclic guanosine monophosphate production was significantly inhibited (to basal concentrations) by L-nitroarginine methyl ester, and prostacyclin production was inhibited by cycloheximide and ibuprofen. Production of thromboxane A(2) by EDVECs was not detected. Endothelin-1 accumulated in the medium, but LPS did not enhance its production. Inducible nitric oxide synthase expression in EDVECs was not detected with the available antibodies, whereas LPS stimulated cyclooxygenase-2 expression in a time- and concentration-dependent manner. CONCLUSIONS AND CLINICAL RELEVANCE: LPS stimulated vasoactive mediator production by equine endothelial cells, which may play a role in LPS-induced digital hypoperfusion.     

Spasmolytic Effect of Caulerpine Involves Blockade of Ca2+ Influx on Guinea Pig Ileum

In this work, we investigated the spasmolytic effect of caulerpine, a bisindole alkaloid isolated from marine algae of the Caulerpa genus, on guinea pig ileum. Our findings indicated that caulerpine inhibited phasic contractions induced by carbachol (IC₅₀ = 7.0 ± 1.9 × 10⁻⁵ M), histamine (IC₅₀ = 1.3 ± 0.3 × 10⁻⁴ M) and serotonin (IC₅₀ = 8.0 ± 1.4 × 10⁻⁵ M) in a non-selective manner. Furthermore, caulerpine concentration-dependently inhibited serotonin-induced cumulative contractions (pD′₂ = 4.48 ± 0.08), shifting the curves to the right with E_max reduction and slope of 2.44 ± 0.21, suggesting a noncompetitive antagonism pseudo-irreversible. The alkaloid also relaxed the ileum pre-contracted by KCl (EC₅₀ = 9.0 ± 0.9 × 10⁻⁵ M) and carbachol (EC₅₀ = 4.6 ± 0.7 × 10⁻⁵ M) in a concentration-dependent manner. This effect was probably due to inhibition of Ca²⁺ influx through voltage-gated calcium channels (Ca_V), since caulerpine slightly inhibited the CaCl₂-induced contractions in depolarizing medium without Ca²⁺, shifting the curves to the right and with E_max reduction. According to these results, the spasmolytic effect of caulerpine on guinea pig ileum seems to involve inhibition of Ca²⁺ influx through Ca_V. However, other mechanisms are not discarded.     

Expression in Escherichia coli of open reading frame gene segments of HTLV-III

Human T-cell lymphotropic virus type III (HTLV-III), the causative agent of the acquired immune deficiency syndrome (AIDS), was recently isolated and its genomic structure analyzed by DNA cloning methods. In the studies reported here a combined cloning and expression system was used to identify HTLV-III encoded peptides that react immunologically with antibodies in sera from AIDS patients. Cloned HTLV-III DNA was sheared into approximately 500-base-pair fragments and inserted into an "open reading frame" expression vector, pMR100. The inserted DNA was expressed in Escherichia coli transformants as a polypeptide fused to the lambda CI protein at its amino terminus and to beta-galactosidase at its carboxyl terminus. Sera from AIDS patients containing antibodies to HTLV-III were then used to screen for immunoreactive fusion proteins. Twenty clones, each specifying a fusion protein strongly reactive with AIDS serum, were identified. DNA sequence analysis indicated that the HTLV-III fragments were derived from the open reading frame DNA segments corresponding to the gag and pol gene coding regions and also the large open reading frame region (env-lor) located near the 3' end of the viral genome.