Characterization of a strawberry late-expressed and fruit-specific peptide methionine sulphoxide reductase

We have cloned and characterized a cDNA clone, called Fapmsr , coding for a putative peptide methionine sulphoxide [Met(O)] reductase (PMSR, EC 1.8.4.6) from strawberry fruits ( Fragaria x ananassa ). This gene is involved in the repair of inactive peptides and proteins caused for the oxidation of methionine residues to Met(O). Expression of the Fapmsr was only detected in the receptacles of red mature fruits and not in young or immature fruits nor in other plant tissues such as flowers, leaves, runners, roots or achenes. Expression of the Fapmsr gene was activated in green immature fruits when achenes were removed from receptacles, and this was prevented by the application of exogenous auxins such as naphthaleneacetic acid. The enzyme produced and purified by cloning the strawberry cDNA in frame with the C-terminal sequence of the glutathione S-transferase gene can reduce free Met(O) to methionine as analysed by reverse phase high performance liquid chromatography. We have also set up a PMSR protection assay that demonstrates that this enzyme can protect in vivo against the damage produced by the addition of H₂O₂.     

A non-invasive technique for the control of intestinal parasites in rhesus macaques (Macaca mulatta).

Control of intestinal parasites is a major concern in maintaining the health and well-being of captive primates. This study established that percutaneous application of ivermectin was as effective as subcutaneous administration for the control of nematodes in captive rhesus macaques. The method is less intrusive than the standard subcutaneous or oral delivery routes and did not adversely affect the health of any of the study subjects.     

Membrane potential-dependent calcium transport in right-side-out plasma membrane vesicles from Zea mays L. roots

Right-side-out plasma membrane vesicles isolated from Zea mays roots were used to study membrane potential (ΔΨ)-dependent Ca²⁺ transport. Membrane potentials were imposed on the vesicles using either K⁺ concentration gradients and valinomycin or SCN concentration gradients, and the size of the imposed ΔΨ was measured with [¹⁴C]tetraphenylphosphonium. Uptake of ⁴⁵Ca²⁺ into the vesicles was stimulated by inside-negative ΔΨ. The rate of transport increased to a maximum at a ΔΨ of about -80 mV and then declined at more negative ΔΨ. When extravesicular Ca²⁺ concentration was varied, uptake was maximal in the range 100–200 μM Ca²⁺. Neither dihydropyridine nor phenylalkylamine Ca²⁺ channel blockers had any effect on Ca²⁺ uptake but 30 μM ruthenium red was completely inhibitory with half maximal inhibition at 10–15 μM ruthenium red. Calcium transport was also inhibited by inorganic cations. Zn²⁺, Gd³⁺ and Mg²⁺ inhibited by a maximum of 30% while La³⁺, Nd³⁺ and Mn²⁺ inhibited by 70%. The inhibitory effects of La³⁺ and Gd³⁺ were additive. Lanthanum-insensitive Ca²⁺ five Ca²⁺ transport was totally inhibited by 80 μM Gd³⁺ and showed maximum activity at a ΔΨ of -60 mV, with less uptake at both higher and lower ΔΨ. Lanthanum and Gd³⁺ also inhibited Ca²⁺ uptake into protoplasts isolated from Zea roots and their individual and combined effects were similar in extent to those observed with plasma membrane vesicles. It is concluded that maize root plasma membrane contains two Ca²⁺-permeable channels that can be distinguished by their susceptibility to inhibition by La³⁺ and Gd³⁺. Both are inhibited by ruthenium red but not by other organic Ca²⁺ channel blockers.     

Rapid chemosensitivity assay with human normal and tumor cells in vitro

Summary Neutral red assay, as an index of cytotoxicity, has been applied to predictive screening of chemotherapeutic agents. Human hepatoma and melanoma tumor cells and normal melanocytes, keratinocytes and fibroblasts were incubated for 2, 24, and 48 h with graded concentrations of cis-platinum (0.1 to 80 μM), doxorubicin (0.01 to 100 μM), and 5-fluorouracil (1 to 1000 μM). Cells were most sensitive after 48 h. Tumor cells, based on 50% toxicity values, were 2–4 times more sensitive than the normal cells, except for cis-platinum, where only melanoma cells, as compared to normal melanocytes, showed a marked difference in cytotoxic response. Methotrexate (1 to 10 μM) toxicity could be reversed in the presence of 100 μM of leucovorin. This sensitive, rapid, and economical assay is suitable for preclinical screening and drug development. This work has been supported, in part, by funds from Schering Corporation, New Jersey, and Chevron Environmental Health Center, Inc., California.     

ISOLATION AND FLOW CYTOMETRIC CHARACTERIZATION OF PROTOPLASTS FROM MARINE MACROALGAE

Protoplasts were isolated from Ulva rigida C. Agardh (Chlorophyta) and two species of Rhodophyta , Gracilariopsis lemaneiformis ( Bory) Dawson, Acleto et Folvik and Gracilaria tenuistipitata Chang et Xia var . liui with minor modifications (the inclusion of 0.01% agarase in the set of cell-wall-degrading enzymes for the two red algae). Flow cytometric characteristics of freshly isolated protoplasts were determined on a FACScan flow cytometer (FC). The most useful parameters for characterizing protoplasts from marine algae were forward angle light scatter (FSC), orange fluorescence (FL2) and red fluorescence (FL3). Protoplasts from all the species were easily distinguishable when their FSC, FL2, and FL3 signals were combined in the bivariate plots FL3 vs. FSC and FL3 us. FL2. Two alternative techniques to help identify protoplasts from debris in the FC computer screen were developed (for FC without sorting capability). Both techniques were based on the ability of new FCs to record time. The first one was based on the induction of rapid changes of cell volume in response to osmotic stress. Only intact protoplasts responded to changes in the osmotic pressure. The second one was based on the uptake and hydrolysis of fluorescein diacetate by intracellular esterases. Viable protoplasts showed a hyperbolic accumulation of fluorescein with time. Semimaximal fluorescein accumulation was attained in 30.5 ± 9.5 s. Debris was easily recognized since, contrary to protoplasts, it did not show a time-dependent accumulation of fluorescein .     

Acidic exopolysaccharide from Bradyrhizobium (Chamaecytisus proliferus)

M. LEON-BARRIOS, A.M. GUTIERREZ-NAVARRO, R. PEREZ-GALDONA, J. DIAZ-SIVERIO. J. TRUJILLO AND J. CORZO. 1992. The exopolysaccharide from a strain of Bradyrhizobium isolated in the Canary Islands was studied. The polysaccharide was found to be acidic and composed of glucose, galactose, mannose and galacturonic acid in a relation 3: 1: 1: 1, respectively. Acetyl was the only acyl substituent detected. Polyacrylamide gel electrophoresis showed that it had a polymeric structure with a variable degree of polymerization. At low ionic strength it aggregated but in EDTA solutions it was resolved as a polydisperse sample by gel exclusion chromatography.