Isolation and culture of rat superior mesenteric artery smooth muscle cells

Summary The structure and function of vascular smooth muscle cells have been extensively investigated with the aid of in vitro culture techniques. The majority of studies have utilized aortic tissue as the source of cells. We present here a method for isolating and culturing smooth muscle cells of the rat superior mesenteric artery, an elasto-muscular vessel that is structurally and functionally different from the aorta. Cells were isolated from partially digested explants and characterized by immunochemical and biochemical techniques. Unlike cultured fibroblasts, the cultured cells stained positive for smooth muscle specific actin. The cells also produced laminin and type IV collagen in culture. This method provides a means for the isolation of large numbers of viable smooth muscle cells from the superior mesenteric artery which can be propagated in culture for in vitro study.     

The conjugative plasmid SLP2 of Streptomyces lividans is a 50 kb linear molecule

The SLP2 plasmid had previously been demonstrated genetically to exist In Streptomyces lividans by its ability to promote conjugation and to elicit‘pocks’on recipient (SLP2^?) cultures, but it had not been physically detected. Using pulsed-field gel electrophoresis, a 50kb linear DNA was isolated from SLP2⁺ but not SLP2^? strains of S. lividans, and from Streptomyces coelicolor and Streptomyces parvulus strains to which SLP2 had been transferred by conjugation or transformation. We conclude that this linear DNA is SLP2. The terminal fragments of SLP2 were cloned. The determined sequences revealed a 44 bp imperfect terminal inverted repeat. The terminal 12 bp sequence of SLP2 was identical to those of two other Streptomyces linear plasmids, pSLA2 and pSCL, and similar to the terminal sequences of another Streptomyces linear plasmid, SCP1. The termini of SLP2 DNA were resistant to digestion by λ exonuclease and ExoIII. A truncated (probably crippled) copy of Tn4811 is present on the plasmid. While the SLP2 plasmid exists as a tree form in the host, a 15.7 kb sequence corresponding to the segment of SLP2 from Tn4811 to the right terminus is also present (at a copy number similar to the free form) elsewhere in the genome of S. lividans. Furthermore, SLP2 is partially homologous to a newly discovered 650 kb linear plasmid in S. parvulus.     

Diol sulfonamides: A potent and novel class of inhibitors of human renin

The syntheses and pharmacological activity of a series of diol sulfonamides which function as inhibitors of human renin are described. The most potent compound in this series, compound 20 (SQ 33,800), is a subnanomolar inhibitor of human renin (IC₅₀ = 0.35 × 10⁻⁹ M).     

Am improved procedure for the isolation of chloroplast DNA fromChlamydomonas reinhardtii

The isolation of chloroplast DNA fromChlamydomonas reinhardtii requires the efficient separation of this AT-rich genome from the GC-rich nuclear genome by density-gradient centrifugation. We describe a simple and efficient method for separating these DNA fractions by using a sodium iodide gradient in combination with the DNA-binding dye, bisbenzimide. The yield of chloroplast DNA is close to the theoretical maximum and the DNA is suitable for restriction enzyme analysis and cloning. This method is applicable to the isolation of AT-rich plastid genomes from other organisms and may be appropriate as a general method for separating species of DNA that differ in their AT/GC ratios. An erratum to this article is available at .     

Effects of temperature and nutrients on microscopic stages of the bull kelp (Nereocystis luetkeana,Phaeophyceae)

Warming ocean temperatures have been linked to kelp forest declines worldwide, and elevated temperatures can act synergistically with other local stressors to exacerbate kelp loss. The bull kelp Nereocystis luetkeana is the primary canopy-forming kelp species in the Salish Sea, where it is declining in areas with elevated summer water temperatures and low nutrient concentrations. To determine the interactive effects of these two stressors on microscopic stages of N. luetkeana, we cultured gametophytes and microscopic sporophytes from seven different Salish Sea populations across seven different temperatures (10–22°C) and two nitrogen concentrations. The thermal tolerance of microscopic gametophytes and sporophytes was similar across populations, and high temperatures were more stressful than low nitrogen levels. Additional nitrogen did not improve gametophyte or sporophyte survival at high temperatures. Gametophyte densities were highest between 10 and 16°C and declined sharply at 18°C, and temperatures of 20 and 22°C were lethal. The window for successful sporophyte production was narrower, peaking at 10–14°C. Across all populations, the warmest temperature at which sporophytes were produced was 16 or 18°C, but sporophyte densities were 78% lower at 16°C and 95% lower at 18°C compared to cooler temperatures. In the field, bottom temperatures revealed that the thermal limits of gametophyte growth (18°C) and sporophyte production (16–18°C) were reached during the summer at multiple sites. Prolonged exposure of bull kelp gametophytes to temperatures of 16°C and above could limit reproduction, and therefore recruitment, of adult kelp sporophytes.     

Impact of Atta colombica Colonies on Understory Vegetation and Light Availability in a Neotropical Forest1

We quantified patterns of vegetation removal and light availability above Atta colombica nests on Barro Colorado Island, Panama. Ants cleared vegetation less than 1 cm in diameter from an area of 77 m², and up to 3 m above ground level. Overall light availability 1.5 m above ground level was 49 percent greater at ant nest sites than at sites in undisturbed understory. These higher light levels fell within the range known to enhance growth of both shade tolerant and pioneer species.     

The homologous tryptophan critical for cytochrome c peroxidase function is not essential for ascorbate peroxidase activity

The crystal structures of ascorbate peroxidase (APX) and cytochrome c peroxidase (CCP) show that the active site structures are nearly identical. Both enzymes contain a His-Asp-Trp catalytic triad in the proximal pocket. The proximal Asp residue hydrogen bonds with both the His proximal heme ligand and the indole ring nitrogen of the proximal Trp. The Trp is stacked parallel to and in contact with the proximal His ligand. This Trp is known to be the site of free radical formation in CCP compound I and also is essential for activity. However, APX forms a porphyrin radical and not a Trp-centered radical, even though the His-Asp-Trp triad structure is the same in both peroxidases. We found that conversion of the proximal Trp to Phe has no effect on APX enzyme activity and that the mutant crystal structure shows that changes in the structure are confined to the site of mutation. This indicates that the paths of electron transfer in CCP and APX are distinctly different. The Trp-to-Phe mutant does alter the stability of the APX compound I porphyrin radical, by a factor of two. Electrostatic calculations and modeling studies show that a potassium cation located about 8?Å from the proximal Trp in APX, but absent in CCP, makes a significant contribution to the stability of a cation Trp radical. This underscores the importance of long-range electrostatic effects in enzyme catalyzed reactions.