RhoA signaling modulates cyclin D1 expression in human lung fibroblasts; implications for idiopathic pulmonary fibrosis

Background

To elucidate further the pathogenesis of sporadic, idiopathic pulmonary arterial hypertension (IPAH) and identify potential therapeutic avenues, differential gene expression in IPAH was examined by suppression subtractive hybridisation (SSH).

Methods

Peripheral lung samples were obtained immediately after removal from patients undergoing lung transplant for IPAH without familial disease, and control tissues consisted of similarly sampled pieces of donor lungs not utilised during transplantation. Pools of lung mRNA from IPAH cases containing plexiform lesions and normal donor lungs were used to generate the tester and driver cDNA libraries, respectively. A subtracted IPAH cDNA library was made by SSH. Clones isolated from this subtracted library were examined for up regulated expression in IPAH using dot blot arrays of positive colony PCR products using both pooled cDNA libraries as probes. Clones verified as being upregulated were sequenced. For two genes the increase in expression was verified by northern blotting and data analysed using Student's unpaired two-tailed t-test.

Results

We present preliminary findings concerning candidate genes upregulated in IPAH. Twenty-seven upregulated genes were identified out of 192 clones examined. Upregulation in individual cases of IPAH was shown by northern blot for tissue inhibitor of metalloproteinase-3 and decorin (P < 0.01) compared with the housekeeping gene glyceraldehydes-3-phosphate dehydrogenase.

Conclusion

Four of the up regulated genes, magic roundabout, hevin, thrombomodulin and sucrose non-fermenting protein-related kinase-1 are expressed specifically by endothelial cells and one, muscleblind-1, by muscle cells, suggesting that they may be associated with plexiform lesions and hypertrophic arterial wall remodelling, respectively.     

ACC oxidase from Carica papaya: Isolation and characterization

Most of the studies done on 1‐aminocyclopropane 1‐carboxylic acid (ACC) oxidase were done in vivo. It is only recently that in vitro studies have been carried out successfully on the enzyme. Here we report on in vitro studies of the enzyme that was isolated from Carica papaya . The enzyme had a K_m of 37 µ M and was inhibited by n ‐propyl gallate (0.240 m M ), sodium dithionite (0.022 m M ), sodium metabisulphite (0.021 m M ) and cobalt sulphate (0.100 m M ). The activity of the enzyme increased with ripening, the enzyme was somewhat labile and activity was lost after 4 days at 14°C; activity was prolonged when the crude homogenate was kept at −15°C. Isolation and purification were achieved with ammonium sulphate precipitation followed by gel‐filtration (Sephadex G 100‐120) and ion‐exchange chromatography (DEAE‐Sephadex). Gel electrophoresis of the purified enzyme gave a single band which corresponded to a molecular mass of 27.5 kDa. The amino acid content of the enzyme showed a relatively high percentage of valine (10.4%). Enzyme activity was enhanced when dithiothreitol (3 m M ) and bicarbonate ion (30 m M ) were added to the assay medium. The production of ethylene from Carica papaya did not require pretreatment of the fruit with ethylene.     

Phosphate uptake into organic compounds in skeletal muscle.

Isolated intact frog muscles were incubated in 32-P-labelled Ringer's solution for various periods of time (30 s-20 h). Labelled compounds were isolated from TCA, methanol-chloroform-water, and water extracts of muscle. Hexosephosphates, phosphocreatine, phosphoenolphyruvate, alpha-glycerol phosphate, adenosine triphosphate, and inorganic phosphate were identified after 30 s, and 4 h incubation. Much more labelling was found after 20 h. The incorporation of 32-P in 30 s into organic phosphate compounds, such as alpha-glycerol phosphate and ATP, showed that immediate esterification of Pi occured on, or just inside, the sarcolemma.     

Fishers' knowledge and seahorse conservation in Brazil

From a conservationist perspective, seahorses are threatened fishes. Concomitantly, from a socioeconomic perspective, they represent a source of income to many fishing communities in developing countries. An integration between these two views requires, among other things, the recognition that seahorse fishers have knowledge and abilities that can assist the implementation of conservation strategies and of management plans for seahorses and their habitats. This paper documents the knowledge held by Brazilian fishers on the biology and ecology of the longsnout seahorse Hippocampus reidi. Its aims were to explore collaborative approaches to seahorse conservation and management in Brazil; to assess fishers' perception of seahorse biology and ecology, in the context evaluating potential management options; to increase fishers' involvement with seahorse conservation in Brazil. Data were obtained through questionnaires and interviews made during field surveys conducted in fishing villages located in the States of Piauí, Ceará, Paraíba, Maranhão, Pernambuco and Pará. We consider the following aspects as positive for the conservation of seahorses and their habitats in Brazil: fishers were willing to dialogue with researchers; although captures and/or trade of brooding seahorses occurred, most interviewees recognized the importance of reproduction to the maintenance of seahorses in the wild (and therefore of their source of income), and expressed concern over population declines; fishers associated the presence of a ventral pouch with reproduction in seahorses (regardless of them knowing which sex bears the pouch), and this may facilitate the construction of collaborative management options designed to eliminate captures of brooding specimens; fishers recognized microhabitats of importance to the maintenance of seahorse wild populations; fishers who kept seahorses in captivity tended to recognize the condtions as poor, and as being a cause of seahorse mortality.     

Pre-incubation of subcellular fractions from rat cerebral cortex inactivates protein phosphorylation.

Pre-incubation of various subcellular fractions from rat brain caused a significant decrease in the phosphorylation of individual polypeptides. The rate and extent of this loss of labelling was not uniform and polypeptides whose phosphorylation was independent of cyclic AMP were primarily affected, whereas substrate availability remained unaltered. It is recommended that pre-incubation effects must be carefully monitored if valid conclusions are to be made about the physiological relevance of changes in protein phosphorylation observed in vitro.     

Improving diagnostic yield of biliary brushings cytology for pancreatic cancer and cholangiocarcinoma

Biliary brushings are currently the best accepted method to obtain a cytological diagnosis of pancreatic cancer or cholangiocarcinoma. The technique has good specificity but poor sensitivity. Two dedicated pathologists reviewed 137 consecutive biliary brushings from 127 patients between February 1997 and February 2000. The ultimate diagnosis was determined by review of radiology, operative diagnosis and patient outcome. The sensitivity, specificity, positive predictive value and negative predictive value of the original results and the review results were calculated and compared. Additional diagnostic categories 'suspicious' and 'atypical possibly benign' were included on review. After review, the sensitivity improved from 49.4% to 89.0% and the specificity remained 100%. The use of the additional diagnostic category 'suspicious' increased the sensitivity to 90.4%, at the expense of a fall of the specificity to 66.7%. We conclude that review by two dedicated pathologists and additional diagnostic categories can improve the diagnostic accuracy of biliary brushings.