Abdominal ultrasound findings in adult patients with cystic fibrosis.

INTRODUCTION AND AIMS: Cystic fibrosis (CF) is a genetic disease whose gastrointestinal compromise mainly involves the pancreas, bile ducts, and liver. Our aim was to analyze abdominal ultrasound findings. METHODS: A retrospective, descriptive study was conducted on adults (patients ≥ 16 years of age) diagnosed with CF, within the time frame of 2006-2019. Clinical and genetic parameters, body mass index, forced expiratory volume in one second, pancreatic insufficiency, CF-related diabetes, cirrhosis secondary to CF, and abdominal ultrasound images were analyzed. RESULTS: Seventy patients, 39 of whom were men (55.8%), had a mean age of 27 years and a mean body mass index of 21.3 ± 2.8 kg/m2 (r: 17-30.9). Forty-seven (67.1%) presented with pancreatic insufficiency, 6 (8.5%) with cirrhosis secondary to CF, and 21 (30%) had CF-related diabetes. Median forced expiratory volume in one second was 47% and the F508del mutation was found in 56.1%. Images of the pancreas: no pathologic findings in 49 (70%), increased echogenicity in 18 (25.7%), and cysts in 3 (4.3%). Gallbladder images: microgallbladder in 3 (4.2%), biliary sludge in 2 (2.8%), gallstones in 7 (10%), and a history of cholecystectomy in 4 (5.8%). Liver and spleen images: no pathologic findings in 47 (67.2%), homogeneous hepatomegaly with splenomegaly in 2 (2.8%), a heterogeneous pattern of the parenchyma in 11 (15.8%), increased echogenicity in 4 (5.7%), and heterogeneous echo patterns, lobulated liver contour, and splenomegaly in 6 (8.5%). CONCLUSION: Abdominal ultrasound is a safe, low-cost technique that enables the identification of some degree of chronic liver and pancreatic diseases, improving the approach and follow-up decisions in adult patients with CF.

Dopamine D1-like receptor activation depolarizes medium spiny neurons of the mouse nucleus accumbens by inhibiting inwardly rectifying K+ currents through a cAMP-dependent protein kinase A-independent mechanism.

Dopamine/cAMP signaling has been reported to mediate behavioral responses related to drug addiction. It also modulates the plasticity and firing properties of medium spiny neurons (MSNs) in the nucleus accumbens (NAc), although the effects of cAMP signaling on the resting membrane potential (RMP) of MSNs has not been specifically defined. In this study, activation of dopamine D1-like receptors (D1Rs) by SKF-38393 elicited membrane depolarization and inward currents in MSNs from the NAc core of 14-17 day-old mice. Similar results were obtained following stimulation of adenylyl cyclase (AC) activity with forskolin or application of exogenous cAMP. Forskolin occluded SKF-38393's effects, thus indicating that D1R action is mediated by AC/cAMP signaling. Accordingly, AC blockade by SQ22536 significantly inhibited the responses to SKF-38393. Effects elicited by D1R stimulation or increased cAMP levels were unaffected by protein kinase A (PKA) or protein kinase C (PKC) blockade and were not mimicked by the Epac agonist, 8CPT-2Me-cAMP. Responses to forskolin were also not significantly modified by cyclic nucleotide-gated (CNG) channel blockade. Forskolin-induced membrane depolarization was associated with increased membrane input resistance. Voltage-clamp experiments revealed that forskolin and SKF-38393 effects were due to inhibition of resting K(+) currents exhibiting inward rectification at hyperpolarized potentials and a reversal potential (around -90 mV) that shifted with the extracellular K(+) concentration. Forskolin and D1R agonist effects were abolished by the inward rectifier K(+) (Kir)-channel blocker, BaCl(2). Collectively, these data suggest that stimulation of postsynaptic D1Rs in MSNs of the NAc core causes membrane depolarization by inhibiting Kir currents. This effect is mediated by AC/cAMP signaling but it is independent on PKA, PKC, Epac and CNG channel activation, suggesting that it may stem from cAMP's direct interaction with Kir channels. D1R/cAMP-mediated excitatory effects may influence the generation of output signals from MSNs by facilitating their transition from the quiescent down-state to the functionally active up-state.

Limitations of Folin assays of foliar phenolics in ecological studies.

We examined the response of the widely used Folin-Denis assay to purified tannins from 16 woody plant species and to three commercial polyphenol preparations often used as standards. The reagent's response to these chemical mixtures differed significantly among sources (tree species, commercial preparations) and sampling dates, even though the mixtures contained the same total dry weight of tannins. Response to commercial standards usually did not resemble response to actual plant tannin and produced estimates that differed from actual concentrations by as much as twofold. Species-based and seasonal differences in polyphenol composition are evidently responsible for these variable results. Reagents that depend on redox reactions, such as the Folin-Denis, do not produce reliable absolute or relative quantification of phenolics when different species or samples from different dates are compared, and use of commercial standards does not resolve this problem.

Thr38 and Ser198 are Pto autophosphorylation sites required for the AvrPto-Pto-mediated hypersensitive response.

The tomato Pto kinase confers resistance to Pseudomonas syringae pv. tomato expressing the AvrPto protein. To elucidate the role of Pto autophosphorylation in disease resistance, eight sites autophosphorylated by Pto in vitro were identified by a combination of HPLC purification of tryptic phosphopeptides, MALDI-TOF/MS analysis and Edman degradation. Mutational analysis of the autophosphorylation sites revealed that Pto residues Thr38 and Ser198 are required for AvrPto-Pto- mediated elicitation of a hypersensitive response in the plant. Thr38, which is the main Pto autophosporylation site and is located outside the kinase catalytic domain, was also required for Pto kinase activity and its physical interaction with AvrPto, the Pti1 kinase and the transcription factor Pti4. Ser198, located in the Pto activation domain, was dispensable for kinase activity and for interaction with AvrPto. However, a mutation at this site resulted in altered Pto interactions with the Pti1 kinase and the Pto interactors of unknown function Pti3 and Pti10. These results suggest that autophosphorylation events at Pto Thr38 and Ser198 are required for signal transduction by Pto and participate in distinct molecular mechanisms.

The major site of the pti1 kinase phosphorylated by the pto kinase is located in the activation domain and is required for pto-pti1 physical interaction.

The Pto and Pti1 serine/threonine protein kinases are key components of the signaling pathway leading to speck disease resistance in tomato. The two kinases physically interact in the yeast two-hybrid system, and Pto specifically phosphorylates Pti1 in vitro. In this study, we identified and characterized the major Pti1 site phosphorylated by Pto. Pto was expressed in Escherichia coli as a maltose-binding fusion protein (MBP-Pto), and used to phosphorylate in vitro a kinase deficient Pti1 protein fused to glutathione S-transferase (GST-Pti1[K96N]). The major phosphopeptide derived from trypsin digestion of phosphorylated GST-Pti1(K96N) was partially purified by reverse-phase HPLC and analyzed by matrix assisted laser desorption/ionization mass spectrometry. Its mass corresponded to phosphopeptide LHSTR, which lies in the Pti1 kinase activation domain at amino acid position 230-234. By phosphoamino acid analysis, Thr233 was determined to be the phosphorylation site of peptide LHSTR. Mutations of Thr233 reduced dramatically Pti1 phosphorylation by MBP-Pto and Pti1 autophosphorylation, providing evidence that the same Pti1 site is involved in the two reactions. Moreover, phosphorylation of Thr233 appeared to be required for Pto-Pti1 physical interaction, as a mutation of this site to alanine, but not to aspartate, abolished the interaction between Pto and Pti1 in the yeast two-hybrid system.

Ca2+ channel inhibition induced by nitric oxide in rat insulinoma RINm5F cells.

The effect of nitric oxide (NO) donors on high-voltage-activated Ca2+ channels in insulin-secreting RINm5F cells was investigated using the patch-clamp technique in the whole-cell configuration. Sodium nitroprusside (SNP, 2-400 microM) induced a dose-dependent reduction in Ba2+ currents with maximal inhibition of 58%. The IC50 for SNP was 45 microM. A different NO donor, (+/-)S-nitroso-N-acetylpenicillamine (SNAP, 500 microM), also produced a 50% decrease in current amplitude. When 200 microM SNP was administered together with the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidozoline-1-oxyl-3-oxide (carboxy-PTIO, 300 microM), the Ba2+ current inhibition was lowered to 7%. Administration of 500 microM 8-bromoguanosine 3':5'-cyclic monophosphate sodium salt (8-Br-cGMP) mimicked the effects of SNP, causing a comparable decrease (56%) in peak-current amplitude. When soluble guanylyl cyclase was blocked by 10 microM 1H-[1,2, 4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ), the inhibitory effect of 200 microM SNP was reduced from 39% to 15%. The SNP-induced current decrease was 36% of controls after the blockade of L-type Ca2+ channels and 30% in the presence of 2.5 microM omega-conotoxin-MVIIC. These data indicate that NO inhibits both L-type and P/Q-type Ca2+ channels in RINm5F cells, probably by an increase in the intracellular levels of cGMP. NO may then significantly influence the Ca2+-dependent release of hormones from secretory cells as well as that of neurotransmitters from nerve terminals.

Erythrocyte engineering for drug delivery and targeting.

A new procedure for the encapsulation of non-diffusible drugs into human erythrocytes was developed. With as little as 50 ml of blood and by using a new apparatus, it was possible to encapsulate a variety of biologically active compounds into erythrocytes in 2 h at room temperature and under blood-banking conditions. The process, which is based on two sequential hypotonic dilutions of washed red cells followed by concentration with a haemofilter and resealing of red cells, allows a 35-50% cell recovery and approx. 30% encapsulation of added drugs. The resulting processed erythrocytes have a normal survival in vivo and can be modified further, with the same apparatus, to increase their recognition by tissue macrophages to perform as a drug-targeting system. The new equipment designed and built for this procedure was named 'Red Cell Loader'.

Biochemical properties of two protein kinases involved in disease resistance signaling in tomato.

In tomato plants, resistance to bacterial speck disease is mediated by a phosphorylation cascade, which is triggered by the specific recognition between the plant serine/threonine protein kinase Pto and the bacterial AvrPto protein. In the present study, we investigated in vitro biochemical properties of Pto, which appears to function as an intracellular receptor for the AvrPto signal molecule. Pto and its downstream effector Pti1, which is also a serine/threonine protein kinase, were expressed in Escherichia coli as maltose-binding protein and glutathione S-transferase fusion proteins, respectively. The two kinases each autophosphorylated at multiple sites as determined by phosphopeptide mapping. In addition, Pto and Pti1 autophosphorylation occurred via an intramolecular mechanism, as their specific activity was not affected by their molar concentration in the assay. Moreover, an active glutathione S-transferase-Pto fusion failed to phosphorylate an inactive maltose-binding protein-Pto(K69Q) fusion excluding an intermolecular mechanism of phosphorylation for Pto. Pti1 phosphorylation by Pto was also characterized and found to occur with a Km of 4.1 microM at sites similar to those autophosphorylated by Pti1. Pto and the product of the recessive allele pto phosphorylated Pti1 at similar sites, as observed by phosphopeptide mapping. This suggests that the inability of the kinase pto to confer resistance to bacterial speck disease in tomato is not caused by altered recognition specificity for Pti1 phosphorylation sites.