Analysis of genetic structure in Melia volkensii (Gurke.) populations using random amplified polymorphic DNA

Melia volkensii (Gurke.) is a popular fast growing agroforestry tree species in the East Africa's arid and semi arid lands (ASALs). The species is valued for its high quality termite resistant timber. In addition, its fruits are eaten by livestock thus making it the species of choice by small-scale farmers. However, the species has been overexploited and information on its existing gene pool is currently lacking. The present work was therefore carried out using random amplified polymorphic DNA (RAPD) markers to assess genetic diversity within and between populations in order to suggest appropriate conservation and management strategies. Eight RAPD primers generated 38 scorable polymorphic bands which were used to estimate genetic distances between populations and for construction of neighbour-joining phenograms. Analysis of Molecular Variance (AMOVA) indicated significant genetic differentiation between populations in the eastern and the coastal regions with 21.1%, (P < 0.0002) of the total variation attributed to differences between these regions. There was a clear split between populations from Eastern and Coastal populations of Kenya. These differences may be due to ecogeographical association with genetic variation and should be conserved to retain the full breadth of genetic variation of the species. Key Words: Melia volkensii, random amplified polymorphic DNA, genetic variation, agroforestry species. African Journal of Biotechnology Vol.3(8) 2004: 421-42

Phosphodiesterase type 4 expression and anti-proliferative effects in human pulmonary artery smooth muscle cells

BACKGROUND: Pulmonary arterial hypertension is a proliferative vascular disease, characterized by aberrant regulation of smooth muscle cell proliferation and apoptosis in distal pulmonary arteries. Prostacyclin (PGI(2)) analogues have anti-proliferative effects on distal human pulmonary artery smooth muscle cells (PASMCs), which are dependent on intracellular cAMP stimulation. We therefore sought to investigate the involvement of the main cAMP-specific enzymes, phosphodiesterase type 4 (PDE4), responsible for cAMP hydrolysis. METHODS: Distal human PASMCs were derived from pulmonary arteries by explant culture (n = 14, passage 3–12). Responses to platelet-derived growth factor-BB (5–10 ng/ml), serum, PGI(2 )analogues (cicaprost, iloprost) and PDE4 inhibitors (roflumilast, rolipram, cilomilast) were determined by measuring cAMP phosphodiesterase activity, intracellular cAMP levels, DNA synthesis, apoptosis (as measured by DNA fragmentation and nuclear condensation) and matrix metalloproteinase-2 and -9 (MMP-2, MMP-9) production. RESULTS: Expression of all four PDE4A-D genes was detected in PASMC isolates. PDE4 contributed to the main proportion (35.9 ± 2.3%, n = 5) of cAMP-specific hydrolytic activity demonstrated in PASMCs, compared to PDE3 (21.5 ± 2.5%), PDE2 (15.8 ± 3.4%) or PDE1 activity (14.5 ± 4.2%). Intracellular cAMP levels were increased by PGI(2 )analogues and further elevated in cells co-treated with roflumilast, rolipram and cilomilast. DNA synthesis was attenuated by 1 μM roflumilast (49 ± 6% inhibition), rolipram (37 ± 6%) and cilomilast (30 ± 4%) and, in the presence of 5 nM cicaprost, these compounds exhibited EC(50 )values of 4.4 (2.6–6.1) nM (Mean and 95% confidence interval), 59 (36–83) nM and 97 (66–130) nM respectively. Roflumilast attenuated cell proliferation and gelatinase (MMP-2 and MMP-9) production and promoted the anti-proliferative effects of PGI(2 )analogues. The cAMP activators iloprost and forskolin also induced apoptosis, whereas roflumilast had no significant effect. CONCLUSION: PDE4 enzymes are expressed in distal human PASMCs and the effects of cAMP-stimulating agents on DNA synthesis, proliferation and MMP production is dependent, at least in part, on PDE4 activity. PDE4 inhibition may provide greater control of cAMP-mediated anti-proliferative effects in human PASMCs and therefore could prove useful as an additional therapy for pulmonary arterial hypertension