Genetic relationships among Mediterranean Pistacia species evaluated by RAPD and AFLP markers

Polymorphisms among Mediterranean basin Pistacia species and accessions within species were assessed by random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) analyses. Twenty-eight Pistacia accessions representing six species from geographically diverse locations in the Mediterranean area were analyzed. With RAPD, a total of 259 DNA fragments were amplified by 27 pre-selected primers, 254 were polymorphic fragments. AFLP analysis with 15 primer sets, produced 954 (93%) polymorphic bands out of a total of 1026. A Mantel test revealed an extremely high correlation (r=0.99) between similarity matrices generated from RAPD and AFLP data sets, indicating that similar results were obtained by the two techniques. Dendrograms constructed from the similarity matrices showed that Pistacia species could be clustered into two groups, one group containing all the #E5/E5#. lentiscus and the second group containing all other accessions. The latter group was divided into two subgroups, one consisting of #E5/E5#. palaestina and #E5/E5#. terebinthus; the other consisting of #E5/E5#. atlantica, #E5/E5#. khinjuk and #E5/E5#. vera. P. vera and P. khinjuk were highly similar, as were P. palaestina and P. terebinthus.     

Nerve growth factor induces 5-HT3 recognition sites in rat pheochromocytoma (PC12) cells

In rat pheochromocytoma (PC12) cells, nerve growth factor (7S NGF) induced the expression of recognition sites that bind the specific 5-HT3 antagonist (S-) [3H]zacopride. Culturing PC12 cells for 8-12 days in the presence of 50 ng/ml NGF increased the density (Bmax) of (S-) [3H]zacopride binding sites in cell membranes (0-100,000 x g fraction) from 0 to 105 fmoles/mg protein. This binding exhibited high affinity for (S-) [3H]zacopride (Kd = 0.8 nM), was specific (greater than 95%), and was inhibited by 5-HT3 compounds with a rank of potency (quipazine greater than ICS 205-930 greater than GR38032F greater than BRL24924 approximately MDL 72222 greater than phenylbiguanide greater than or equal to serotonin greater than 2-methyl-serotonin greater than metoclopramide) which was distinct from neuroblastoma cells. Thus, NGF-differentiated PC12 cells possess a 5-HT3 receptor and should be useful to investigate its regulation and biochemical mechanism of action.     

Identification of an estrogen-binding protein in Pseudomonas aeruginosa.

A constitutive estrogen-binding protein (EBP) has been identified in the cytosol of Pseudomonas aeruginosa, a Gram-negative bacterium. All 14 strains tested contained the EBP. Estradiol binding was rapid and maximal binding occurred by 90 min at 0 degrees C. Dissociation of estradiol from the binding protein occurred at a rate of 4.6 fmol/min with a t1/2 of 42 min. EBP binding was destroyed by protease treatment and at high temperature. Sodium molybdate had no effect on binding. The Kd determined by Scatchard analysis was 3.9 nM and the Bmax was 323 fmol/mg protein. The EBP sedimented at 8.9 S on sucrose density gradients. The presence of 0.4 M KCl increased estradiol binding 6-fold but did not cause a shift in the sedimentation value. Gel filtration of the native protein gave an estimated molecular weight of 215,000 and a Stokes radius of 50.2 A. Steroid binding specificity, in order of decreasing affinity, was estradiol, estrone, dihydrotestosterone, estriol, testosterone, progesterone and promegestone. Other steroid hormones tested did not compete for estradiol binding. Identification of an EBP in a bacterium allows a comparative analysis of other steroid-binding proteins in unicellular microorganisms.     

Purification and characterization of a secreted recombinant phosphotriesterase (parathion hydrolase) from Streptomyces lividans.

A heterologous phosphotriesterase (parathion hydrolase), previously cloned from a Flavobacterium species into Streptomyces lividans, was secreted at high levels and purified to homogeneity. N-terminal analysis revealed that it had been processed in the same manner as the native membrane-bound Flavobacterium hydrolase. The enzyme consisted of a single polypeptide with an apparent molecular weight of 35,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Substrate specificity studies showed Kms of 68 microM for parathion, 46 microM for O-ethyl O-p-nitrophenyl phenylphosphonothioate, 599 microM for methyl parathion, and 357 microM for p-nitrophenyl ethyl(phenyl)phosphinate. Temperature and pH optima were 45 degrees C and 9.0, respectively. The purified enzyme was inhibited by 1 mM dithiothreitol and 1 mM CuSO4. After chelation and inactivation by o-phenanthroline, however, activity could be partially restored by 1 mM CuCl or 1 mM CuSO4. The results showed that the purified recombinant parathion hydrolase has the same characteristics as the native Flavobacterium hydrolase. This system provides a source of milligram quantities of parathion hydrolase for future structural and mechanism studies and has the potential to be used in toxic waste treatment strategies.     

Infection of central nervous system cells by ecotropic murine leukemia virus in C58 and AKR mice and in in utero-infected CE/J mice predisposes mice to paralytic infection by lactate dehydrogenase-elevating virus.

Certain mouse strains, such as AKR and C58, which possess N-tropic, ecotropic murine leukemia virus (MuLV) proviruses and are homozygous at the Fv-1n locus are specifically susceptible to paralytic infection (age-dependent poliomyelitis [ADPM]) by lactate dehydrogenase-elevating virus (LDV). Our results provide an explanation for this genetic linkage and directly prove that ecotropic MuLV infection of spinal cord cells is responsible for rendering anterior horn neurons susceptible to cytocidal LDV infection, which is the cause of the paralytic disease. Northern (RNA) blot hybridization of total tissue RNA and in situ hybridization of tissue sections demonstrated that only mice harboring central nervous system (CNS) cells that expressed ecotropic MuLV were susceptible to ADPM. Our evidence indicates that the ecotropic MuLV RNA is transcribed in CNS cells from ecotropic MuLV proviruses that have been acquired by infection with exogenous ecotropic MuLV, probably during embryogenesis, the time when germ line proviruses in AKR and C58 mice first become activated. In young mice, MuLV RNA-containing cells were found exclusively in white-matter tracts and therefore were glial cells. An increase in the ADPM susceptibility of the mice with advancing age correlated with the presence of an increased number of ecotropic MuLV RNA-containing cells in the spinal cords which, in turn, correlated with an increase in the number of unmethylated proviruses in the DNA extracted from spinal cords. Studies with AKXD recombinant inbred strains showed that possession of a single replication-competent ecotropic MuLV provirus (emv-11) by Fv-1n/n mice was sufficient to result in ecotropic MuLV infection of CNS cells and ADPM susceptibility. In contrast, no ecotropic MuLV RNA-positive cells were present in the CNSs of mice carrying defective ecotropic MuLV proviruses (emv-3 or emv-13) or in which ecotropic MuLV replication was blocked by the Fv-1n/b or Fv-1b/b phenotype. Such mice were resistant to paralytic LDV infection. In utero infection of CE/J mice, which are devoid of any endogenous ecotropic MuLVs, with the infectious clone of emv-11 (AKR-623) resulted in the infection of CNS cells, and the mice became ADPM susceptible, whereas littermates that had not become infected with ecotropic MuLV remained ADPM resistant.