No abstract

No abstract

Our results suggest that neointimal cells in the rat balloon injury model mostly derive from pre-existing vascular cells and that only a small population of those cells come from BM-derived progenitors.

Nicotine acts through its receptors in VSMC to activate the ERK-Egr-1 signaling cascade that induces cell proliferation and exacerbates post-injury neointimal development.

A cDNA from Penicillium minioluteum HI-4 encoding a dextranase (1,6-a-glucan hydrolase, EC 3.2.1 .l 1) was isolated and characterized. cDNA clones corresponding to genes expressed in dextran-induced cultures were identified by differential hybridization. Southern hybridization and restriction mapping analysis of selected clones revealed four different groups of cDNAs. The dextranase cDNA was identified after expressing a cDNA fragment from each of the isolated groups of cDNA clones in the Escherichia coli T7 system. The expression of a 2 kb cDNA fragment in E. coli led to the production of a 67 kDa protein which was recognized by an anti-dextranase polyclonal antibody. The cDNA contains 2109 bp plus a poly(A) tail, coding for a protein of 608 amino acids, including 20 N-terminal amino acid residues which might correspond to a signal peptide. There was 29% sequence identity between the P. minioluteum dextranase and the dextranase from Arthrobacter sp. CB-8.

Alteration of VSMC (vascular smooth-muscle cell) physiology is associated with the development of atherosclerosis and restenosis. We hypothesize that aging up-regulates the expression of p16 INK4a in VSMCs, which may increase the susceptibility of blood vessels to vascular occlusive diseases. Aortic VSMCs were obtained from young and aged mice. Cells from aged mice grew more slowly than those from their younger counterparts. Progression of cell cycle in response to serum stimulation was significantly inhibited in those cells with aging, as determined by FACS after propidium iodide staining. A significant up-regulation of p16 INK4a (2.5-fold, P=0.0012) was found in VSMC from aged animals using gene arrays. The up-regulation of this gene was further confirmed by quantitative RT-PCR (reverse transcription-PCR) and Western-blot experiments. Immunostaining for p16 INK4a confirmed that aortas from aged mice contained more p16 INK4a+ SMA (smooth-muscle cell actin)+ cells than aortas from young animals (26.79+/-2.45 versus 7.06+/-1.44, P=0.00027, n=4). In conclusion, we have shown that aging up-regulates the expression of p16 INK4a in VSMC in both cultures and arteries. The increase in p16 INK4a in the vasculature with aging may modify VSMC's response to post-injury stress and therefore accelerate the development of age-related cardiovascular diseases.

Presence of subtypes of voltage-dependent Ca channels was investigated in young and old human red cells, employing immunological and flux-kinetics methods. Western blots showed specific reaction toward polyclonal rabbit antibodies raised against a highly conserved residue of alpha1 subunit of high-voltage activated Ca channels (pan alpha1) and against conserved residues of alpha1C and alpha1E subunits. No specific reaction was detected with antibodies against conserved residues of alpha1A, alpha1B, or alpha1D subunits. Only a single band (approx 260 kDa) was revealed on anti-pan alpha1 or anti-alpha1E blots, whereas two bands (200 and 230 kDa) were detected by anti-alpha1C exposure. Blots from old cells always showed diminished band intensity. Channel activity was assessed by studying the effect of voltage-dependent Ca channels blockers under conditions likely to alter the red cell membrane potential, through incubation in media of different composition. In a 150 mM NaCl + 5 mM KCl medium, blockers of L-, R-, and Q-type caused a 15-50% reduction of 45Ca influx into cells, which had the Ca pump inactivated by either exhaustive adenosine triphosphate depletion or presence of vanadate plus substrates. Additionally, some P/Qand N-type blockers also reduced Ca influx to various extents (25-60%). Old cells were generally insensitive to L-type but not to non-L-type blockers. Raising external K to about 70-80 mM reduced by 50-100% inhibition by L-type blockers. Incubation in a gluconate medium containing 150 mM Na+5 mM K practically abolished the action of L-type blockers, but only slightly reducing that by non-L-type. The results clearly demonstrate presence of L- and R-type Ca channels, apparently occurring in different functional states in young and old cells. Other non-L-type channels were also demonstrated only by pharmacological means. A possible physiological role for these channels is discussed.

The soluble guanylyl cyclases (sGC), the receptor for nitric oxide, are heterodimers consisting of an α‐ and β‐subunit. This study aimed to investigate the translational mechanism of the sGC β2‐subunit. Two mRNA species for sGC β2 were isolated from human kidney. These transcripts had dissimilar 5′‐untranslated regions (5′‐UTRs). The most abundant sGC β2 mRNA showed numerous upstream open reading frames (ORFs) and stable secondary structures that inhibited in vivo and in vitro translation. To evaluate whether these 5′‐UTRs harbored an internal ribosome entry site (IRES) that allows translation by an alternative mechanism, we inserted these regions between the two luciferase genes of a bicistronic vector. Transfection of those genetic constructs into HeLa cells demonstrated that both sGC β2 leaders had IRES activity in a cell‐type dependent manner. Finally, the secondary structural model of the sGC β2 5′‐UTR predicts a Y‐type pseudoknot that characterizes the IRES of cellular mRNAs. In conclusion, our findings suggest that sGC β2 5′‐UTRs have IRES activity that may permit sGC β2 expression under conditions that are not optimal for scanning‐dependent translation.