Regulation of acetyl-CoA:1-alkyl-sn-glycero-3-phosphocholine O2-acetyltransferase (lyso-PAF-acetyltransferase) in exocrine glands. Evidence for an activation via phosphorylation by calcium/calmodulin-dependent protein kinase

Stimulation of secretion in guinea pig exocrine cells is associated with an enhanced synthesis in these cells of 1-O-alkyl-2-sn-acetyl-glycero-3-phosphocholines (PAF) from 1-O-alkyl-sn-glycero-3-phosphocholine (lyso-PAF) (S?ling, H-D., and Fest, W. (1986) J. Biol. Chem. 261, 13916-13922). This results from a stimulation of the activity of lyso-1-alkylglycerophosphocholine acetyltransferase (EC 2.3.1.67). Here we have analyzed the effects of various agonists on the activity of this enzyme in guinea pig parotid gland microsomes. Carbamoylcholine leads within less than 30 s to a 2- to 4-fold activation of lyso-PAF-acetyltransferase, which persists after solubilization of the microsomal enzyme with octyl glucoside. The calcium ionophore A23187 has a similar though smaller effect. Neither isoproterenol (2 X 10(-5) M), which stimulates exocytosis more than carbachol, nor phorbol ester significantly affected lyso-PAF-acetyltransferase activity. Incubation of microsomes from unstimulated parotid gland acini with cAMP-dependent and calcium/calmodulin-dependent protein kinase resulted in a 4-fold and 2.9-fold activation of lyso-PAF-acetyltransferase activity, respectively. Protein kinase C had no significant effect. Activation with calcium/calmodulin-dependent protein kinase was inhibited by 40 microM trifluoperazine. When microsomes from carbachol-stimulated glands were used, in vitro activation of the enzyme by calcium/calmodulin-dependent protein kinase was almost abolished. Protein phosphatase 2A in vitro strongly reduced lyso-PAF-acetyltransferase activity in microsomes from both stimulated and unstimulated glands, whereas alkaline phosphatase and protein phosphatase 1 had only small effects. Following treatment with protein phosphatase 2A, enzyme activity in microsomes from stimulated glands could be enhanced more than 8-fold by subsequent incubation with calcium/calmodulin-dependent protein kinase. Although unsuccessful attempts have made it impossible so far to demonstrate directly the incorporation of phosphate into lyso-PAF-acetyltransferase, the results reported here strongly suggest that the enzyme in exocrine cells is regulated by phosphorylation-dephosphorylation and that a calcium/calmodulin-dependent protein kinase is responsible for the activation of the enzyme and type-2 protein phosphatases for its inactivation.     

POLYMORPHISM IN THE DESMID XANTHIDIUM REGULARE AND ITS TAXONOMIC IMPLICATIONS

This paper refers to a case of polymorphism in the desmid genus Xanthidium Ehr. It is based on material from Lake Dais Irmaios, the main body of water in the Zoological and Botanical Garden in Recife, Pernambuco, northeastern Brazil, collected at 4 different times of the year during 1967 and 1968. A detailed examination of almost 1300 specimens showed an enormous variety in form of Xanthidium regulare Nordst., X. fragile Borge, and X. pseudoregulare Borge, thus allowing the authors to draw the following conclusions: (1) the name X. regulare Nordst. should be retained until further and more detailed studies on form variation within the species are available; (2) the names X. regulare Nordst. var. asteptum Nordst. in Borge, X. regulare Nordst. var. sexangulare Grönbl., X. regulare Nordst. var. sexangulare Grönbl. f. robustior Grönbl., X. fragile Borge, X. fragile Borge forma, and X. fragile Borge var. depauperatum Borge should be considered synonymous, all referring to a single variety of X. regulare Nordst., var. asteptum Nordst. in Borge emend. C. Bic. & L. M. Carv.; (3) X. pseudoregulare Borge must be treated as a variety of X. regulare Nordst. and must be called X. regulare Nordst. var. pseudoregulare (Barge) C. Bic. & L. M. Carv. Finally, a key is given to the 3 varieties of X. regulare Nordst. proposed in the present paper.     

Pseudomonas aeruginosa acid phosphatase and cholinesterase induced by choline and its metabolic derivatives may contain a similar anionic peripheral site

Summary Different compounds derived from choline, and obtained by demethylation or by oxidation of the primary alcohol group with subsequent N-demethylation, were tested as inducer agents of acid phosphatase and cholinesterase in Ps. aeruginosa. It was found that betaine and dimethylglycine were the most effective inducers of both enzyme activities. These metabolites including choline itself, were not inducers of acid phosphatase and cholinesterase in other Gram-negative bacteria such as: Escherichia coli, Salmonella typhimurium, Shigella flexneri, Enterobacter liquefacciens and Proteus mirabilis. The acid phosphatase activities found in these bacteria were not inhibited in vitro by choline, betaine and phosphorylcholine. From these results it may be concluded that the acid phosphatase activity from Ps. aeruginosa is different from the same activity observed in the other bacteria. In addition, it is also shown that Ps. aeruginosa acid phosphatase and cholinesterase were inhibited by a number of compounds containing a positively charged amino group, with methyl or ethyl groups bound to it. These results seem to confirm that Ps. aeruginosa acid phosphatase and cholinesterase may contain a similar anionic site.     

One mutation,three phenotypes: novel metabolic insights on MELAS,MIDD and myopathy caused by the m.3243A > G mutation

Metabolomics - Studies investigating crop resistance to abiotic and biotic stress have largely focused on plant responses to singular forms of stress and individual biochemical pathways that only...     

Mesenchymal Stem Cells (MSC) Prevented the Progression of Renovascular Hypertension,Improved Renal Function and Architecture

Renovascular hypertension induced by 2 Kidney-1 Clip (2K-1C) is a renin-angiotensin-system (RAS)-dependent model, leading to renal vascular rarefaction and renal failure. RAS inhibitors are not able to reduce arterial pressure (AP) and/or preserve the renal function, and thus, alternative therapies are needed. Three weeks after left renal artery occlusion, fluorescently tagged mesenchymal stem cells (MSC) (2×10⁵ cells/animal) were injected weekly into the tail vein in 2K-1C hypertensive rats. Flow cytometry showed labeled MSC in the cortex and medulla of the clipped kidney. MSC prevented a further increase in the AP, significantly reduced proteinuria and decreased sympathetic hyperactivity in 2K-1C rats. Renal function parameters were unchanged, except for an increase in urinary volume observed in 2K-1C rats, which was not corrected by MSC. The treatment improved the morphology and decreased the fibrotic areas in the clipped kidney and also significantly reduced renal vascular rarefaction typical of 2K-1C model. Expression levels of IL-1β, TNF-α angiotensinogen, ACE, and Ang II receptor AT₁ were elevated, whereas AT₂ levels were decreased in the medulla of the clipped kidney. MSC normalized these expression levels. In conclusion, MSC therapy in the 2K-1C model (i) prevented the progressive increase of AP, (ii) improved renal morphology and microvascular rarefaction, (iii) reduced fibrosis, proteinuria and inflammatory cytokines, (iv) suppressed the intrarenal RAS, iv) decreased sympathetic hyperactivity in anesthetized animals and v) MSC were detected at the CNS suggesting that the cells crossed the blood-brain barrier. This therapy may be a promising strategy to treat renovascular hypertension and its renal consequences in the near future.     

Widening the antimicrobial spectrum of esters of bicyclic amines: In vitro effect on gram-positive Streptococcus pneumoniae and gram-negative non-typeable Haemophilus influenzae biofilms

Antibiotic resistance is a global current threat of increasing importance. Moreover, biofilms represent a medical challenge since the inherent antibiotic resistance of their producers demands the use of high doses of antibiotics over prolonged periods. Frequently, these therapeutic measures fail, contributing to bacterial persistence, therefore demanding the development of novel antimicrobials. Esters of bicyclic amines (EBAs), which are strong inhibitors of Streptococcus pneumoniae growth, were initially designed as inhibitors of pneumococcal choline-binding proteins on the basis of their structural analogy to the choline residues in the cell wall. However, instead of mimicking the characteristic cell chaining phenotype caused by exogenously added choline on planktonic cultures of pneumococcal cells, EBAs showed an unexpected lytic activity. In this work we demonstrate that EBAs display a second, and even more important, function as cell membrane destabilizers. We then assayed the inhibitory and disintegrating activity of these molecules on pneumococcal biofilms. The selected compound (EBA 31) produced the highest effect on S. pneumoniae (encapsulated and non-encapsulated) biofilms at very low concentrations. EBA 31 was also effective on mixed biofilms of non-encapsulated S. pneumoniae plus non-typeable Haemophilus influenzae, two pathogens frequently forming a self-produced biofilm in the human nasopharynx. These results support the role of EBAs as a promising alternative for the development of novel, broad-range antimicrobial drugs encompassing both Gram-positive and Gram-negative pathogens.     

Development of α4 integrin DNA aptamer as a potential therapeutic tool for multiple sclerosis

One of the most important molecules for multiple sclerosis pathogenesis is α4 integrin, which is responsible for autoreactive leukocytes migration into the brain. The monoclonal antibody, natalizumab, was introduced to market for blocking the extravasation of autoreactive leukocytes via inhibition of α4 integrin. However, the disadvantages of antibodies provided a suitable background for other agents to be replaced with antibodies. Considering the profound advantages of aptamers over antibodies, aptamer isolation against α4 integrin was intended in the current study. The α4 integrin-specific aptamers were selected using cell-systematic evolution of ligands by exponential enrichment (SELEX) method with human embryonic kidney (HEK)-293T overexpressing α4 integrin and HEK-293T as target and control cells, respectively. Evaluation of selected aptamer was performed through flow cytometric analysis. The selected clones were then sequenced and analyzed for any possible secondary structure and affinity. The results of this study led to isolation of 13 different single-stranded DNA clones in 11 rounds of selection which were categorized to three clusters based on common structural motifs and the equilibrium dissociation constant (K _d) of the most stable structure was calculated. The evaluation of SELEX progress showed growth in aptamer affinity with increasing of the number of cycles. Taken together, the findings of this study demonstrated the isolation of α4-specific single-stranded DNA aptamers with suitable affinity for ligand, which can further be replaced with natalizumab.     

The C-type natriuretic peptide precursor of snake brain contains highly specific inhibitors of the angiotensin-converting enzyme

The bradykinin-potentiating peptides from Bothrops jararaca venom are the most potent natural inhibitors of the angiotensin-converting enzyme. The biochemical and biological features of these peptides were crucial to demonstrate the pivotal role of the angiotensin-converting enzyme in blood pressure regulation. In the present study, seven bradykinin-potentiating peptides were identified within the C-type natriuretic peptide precursor cloned from snake brain. The bradykinin-potentiating peptides deduced from the B. jararaca brain precursor are strong in vitro inhibitors of the angiotensin-converting enzyme (nanomolar range), and also potentiate the bradykinin effects in ex vivo and in vivo experiments. Two of these peptides are novel bradykinin-potentiating peptides, one of which displays high specificity toward the N-domain active site of the somatic angiotensin-converting enzyme. In situ hybridization studies revealed the presence of the bradykinin-potentiating peptides precursor mRNAs in distinct regions of the B. jararaca brain, such as the ventromedial hypothalamus, the paraventricular nuclei, the paraventricular organ, and the subcommissural organ. The biochemical and pharmacological properties of the brain bradykinin-potentiating peptides, their presence within the neuroendocrine regulator C-type natriuretic peptide precursor, and their expression in regions of the snake brain correlated to neuroendocrine functions, strongly suggest that these peptides belong to a novel class of endogenous vasoactive peptides.     

Mitochondrial ATP-sensitive K+ channel opening decreases reactive oxygen species generation

Mitochondrial ATP-sensitive K(+) channel (mitoK(ATP)) opening was shown previously to slightly increase respiration and decrease the membrane potential by stimulating K(+) cycling across the inner membrane. Here we show that mitoK(ATP) opening reduces reactive oxygen species generation in heart, liver and brain mitochondria. Decreased H(2)O(2) release is observed when mitoK(ATP) is active both with respiration stimulated by oxidative phosphorylation and when ATP synthesis is inhibited. In addition, decreased H(2)O(2) release is observed when mitochondrial Delta pH is enhanced, an effect expected to occur when mitoK(ATP) is open. We conclude that mitoK(ATP) is an effective pathway to trigger mild uncoupling, preventing reactive oxygen species release.