Metformin, an antidiabetic agent, potentiates insulin action and reduces insulin resistance. We examined the antihypertensive effects and vascular effects of metformin in spontaneously hypertensive rats (SHR). Wistar-Kyoto normotensive (WKY) and SHR were injected with metformin (100 mg/kg) or saline subcutaneously twice daily for 4 weeks. Blood pressure was recorded by a tail-cuff plethesmographic method. Metformin treatment significantly attenuated (P < .05) the increase in blood pressure in metformin treated SHR versus untreated control SHR. At the end of the experimental period of 4 weeks, metformin-treated SHR had a mean blood pressure that was 34 mm lower than that of untreated SHR. Metformin treatment had no significant effect on blood pressure in WKY rats. Treatment of SHR aortic smooth muscle (SM) cells with metformin (2 micrograms/mL) for 24 h significantly decreased (P < .05) arginine vasopressin- and thrombin- stimulated increase in [Ca2+]i. However, metformin treatment did not have a significant effect on the basal [Ca+]i. Incubation of SHR aortic SM cells with OH-L-arginine (25 to 100 mumol/L) for 24 h increased nitrite production in a dose dependent manner. Metformin (5 micrograms/mL) treatment of SM cells increased nitrite production at all concentrations of OH-L-arginine; however, differences were significant (P < .05) only at 25 and 50 mumol/L OH-L-arginine. These results suggest that metformin may be decreasing arterial pressure in the SHR, at least in part, by attenuating the agonist-stimulated [Ca2+]i response in SHR vascular smooth muscle cells.

Epidemiological evidence and estrogen replacement studies suggest that estrogen has a protective effect on the cardiovascular system against coronary artery disease. Vascular smooth muscle (VSM) cell replication has been shown to play a causative role in the pathogenesis of atherosclerosis. Therefore, in this study, we investigated the effect of chronic treatment of cultured guinea pig coronary artery VSM cells with physiological concentrations of 17beta-estradiol (E2) on thymidine incorporation, cell proliferation, and bradykinin-stimulated cytosolic calcium concentration ([Ca2+]i). Bradykinin at physiological concentrations causes contraction of endothelium-denuded guinea pig coronary artery rings in a concentration-dependent manner. VSM cells were first treated with low doses of E2 (10 pg/ml) for 1-2 days followed by treatment for 4-6 days with 50 pg/ml of E2, a concentration similar to that found in pregnancy. Using these protocols, we consistently observed the presence of E2-receptor mRNA in VSM cells by a ribonuclease protection assay. Fetal calf serum-stimulated [3H]thymidine incorporation was significantly reduced (P < 0.05) in E2-treated cells compared with untreated control cells. Similarly, E2 treatment significantly inhibited fetal calf serum-stimulated VSM cell proliferation compared with untreated control cells (P < 0.05). We also tested the hypothesis that E2 treatment attenuates agonist-stimulated [Ca2+]i in VSM cells because acute E2 treatment has been shown to produce relaxation of precontracted isolated coronary artery preparations. E2 treatment of VSM cells resulted in a significant decrease in bradykinin-stimulated [Ca2+]i compared with untreated cells (P < 0.05). In conclusion, our data demonstrate that estrogen at physiological concentrations directly regulates coronary VSM cell function.

A gene for a ribosomal protein, rps3, was amplified by PCR and sequenced from representatives of the class Mollicutes. Alignments of the deduced amino acid sequences allowed the construction of a phylogeny that is consistent with the phylogenetic trees created from 5s and 16s rRNA comparisons, including the position of the former Acholeplasma florum on the Mycoplusma branch, rather than with the classical Acholeplasmataceae. Additional confirmation of the phylogeny comes from the deduction that the UGA triplet encodes tryptophan in the rps3 gene from Mesoplasmuflorum, as it does in the mycoplasmas and spiroplasmas. The sequence data from Acholeplasma axanthum 743 and Acholeplusma sp. strain 5233 allow refinements to the phylogenetic tree within the Acholeplusmutuceae, providing evidence that the sterol requirement of Anueroplusma abactoclasticum (order Anaeroplasmatales) is a derived trait. It was also evident that the nonhelical plant-pathogenic members of the class Mollicutes, referred to as mycoplasmalike organisms or phytoplasmas, are more closely related to the true acholeplasmas (Acholeplusma laidlawii and strain 5233) than to other members of the Mollicutes.The placement of certain non-sterol-requiring, cell wallless bacteria from plants and insects into the family Acholeplasmataceae of the class Mollicutes was brought into question by 16s rRNA sequence data from a large number of these species (13,31,34). The 16s rRNA data indicated that two species that were classified as acholeplasmas, Acholeplasma frorum and the closely related Acholeplasma entomophilum, were phylogenetically closer to members of the Mycoplasmataceae and Spiroplasmataceae than to the other members of the Acholeplasmataceae. These data, together with a reassessment of nutritional requirements, led to the suggestion that they be renamed Mesoplasma florum and Mesoplasma entomophilum and, along with other closely related members of the Mollicutes, including all species of Spiroplasma, be placed in a new order, the Entomoplasmatales (31).The phylogenetic trees constructed from 5s and 16s rRNA data (27, 34) show that the Anaeroplasmataceae are more closely related to the classical Acholeplasmataceae than to the other mollicutes, even though species of Anaeroplasma require sterols, as do all species of Mycoplasma and Spiroplasma. The 16s rRNA sequence data from the plantpathogenic mycoplasmalike organisms (MLOs) indicated a phylogenetic position between the Acholeplasmatales and the Anaeroplasmatales (9, 10). Considering these relationships, the possibility arose that the difficulty in developing an in vitro culture medium for MLOs could be due to an oxygen-sensitive metabolism in MLOs or to incorrect guesses about their sterol requirements. To provide supplemental data to address the question of phylogeny of the MLOs, we have obtained sequence data from a gene for a ribosomal protein, rps3, for which comparable data were already available from Mycoplasma capricolum (19), Acholeplasma laidlawii (ll), and an MLO (11). In this report, * Corres...

Meristems from 25 -90-year-old oak (Quercus robur L and Q. petraea Matt.) trees and seed embryos were pretreated with polyvinyl pyrrolidone, ascorbic acid, cysteine and citric acid solutions. Tissues were cultured mostly on a WPM medium supplemented with different combinations and concentrations of growth regulators. All the different pretreatments showed a positive effect against the otherwise very rapid and harmful browning of the explants, but ascorbic acid (100 mg dm -3) proved to be the most effective. Shooting was induced from seed embryos and meristems originating from adult trees. Rooted plantlets were obtained from explants of seed embryos.

Most research involving phloem proteins is done with phloem exudates, which are not easily obtained from many plants. We report here on the use of tissue cultures to study phloem proteins. Monoclonal antibodies against the filamentous phloem protein, P‐protein, were made by injecting mice with a phloem‐enriched fraction isolated from Streptanthus tortuosus callus grown on a medium that stimulates the differentiation of xylem and phloem (phloem[+] cultures). Monoclonal antibodies specific for P‐protein were identified by incubating free‐hand stem sections of S. tortuosus in hybridoma supernatants, then in a goat anti‐mouse antibody conjugated to fluorescein isothiocyanate (FITC), and observing the FITC under an epifluorescence microscope. Antibodies specific for P‐protein in stem sections were used to probe nitrocellulose blots of polyacrylamide gels separating proteins isolated from both phloem(+) and phloem(‐) tissue cultures. Immunoblots were incubated overnight in hybridoma supernatants followed by a secondary antibody conjugated to alkaline phosphatase. Three monoclonal antibodies—RS21, RS22, and RS23—bound to an 89‐kD band in the phloem(+) lanes but failed to bind to any proteins in the phloem(—) lanes. In leaf sections of Arabidopsis thaliana processed by freeze‐substitution, a mixture of RS21 and RS22 bound to the P‐protein filaments in sieve elements, but not to any proteins in adjacent cells. A control antibody specific for tubulin did not bind to the P‐protein filaments.