Phylogenetic characterization of ineffective Frankia in Alnus glutinosa (L.) Gaertn. nodules from wetland soil inoculants

Ineffective Frankia endophytes were retrieved from various wet soils by using Alnus glutinosa clones as trapping plants. No pure cultures could be isolated from these ineffective nodules. Therefore, the phylogenetic position of these endophytes was determined by sequence analysis of cloned PCR products of bacterial 16S rDNA, derived from nodules. The results showed that all nodule endophytes belong to a hitherto undescribed cluster of the Frankia phylogenetic tree. The position of these uncultured ineffective Frankia nodule endophytes is different from that of the ineffective Frankia isolates derived from A. glutinosa nodules, even when originating from the same geographical location. This suggests a bias in current isolation techniques.     

Molecular evolution of a portion of the mitochondrial 16S ribosomal gene region in scleractinian corals

Relationships among families and suborders of scleractinian corals are poorly understood because of difficulties 1) in making inferences about the evolution of the morphological characters used in coral taxonomy and 2) in interpreting their 240-million-year fossil record. Here we describe patterns of molecular evolution in a segment of the mitochondrial (mt) 16S ribosomal gene from taxa of 14 families of corals and the use of this gene segment in a phylogenetic analysis of relationships within the order. We show that sequences obtained from scleractinians are homologous to other metazoan 16S ribosomal sequences and fall into two distinct clades defined by size of the amplified gene product. Comparisons of sequences from the two clades demonstrate that both sets of sequences are evolving under similar evolutionary constraints: they do not differ in nucleotide composition, numbers of transition and transversion substitutions, spatial patterns of substitutions, or in rates of divergence. The characteristics and patterns observed in these sequences as well as the secondary structures, are similar to those observed in mt 16S ribosomal DNA sequences from other taxa. Phylogenetic analysis of these sequences shows that they are useful for evaluating relationships within the order. The hypothesis generated from this analysis differs from traditional hypotheses for evolutionary relationships among the Scleractinia and suggests that a reevaluation of evolutionary affinities in the order is needed. Received: 4 September 1996 / Accepted: 7 April 1997     

A 250-kilodalton cellular protein is induced by progestins in two human breast cancer cell lines MCF7 and T47D

We have studied the effect of R5020, a synthetic progestin, on the biosynthesis of cellular proteins extracted from the MCF7 and T47D human breast cancer cells, using gel electrophoresis. R5020 stimulates the synthesis, as measured after [35S]-methionine labelling, and the accumulation, as shown by silver staining, of a protein of molecular weight approximately equal to 250,000. The increase of the labelled 250-kilodalton protein was rapid (3 hours) and after 3 days this protein represented approximately equal to 6% of the total cellular proteins (approximately equal to 1 microgram/150,000 cells). The induction of the 250-kilodalton protein was obtained by physiologically active concentrations of several progestins and high concentrations of 5 alpha-dihydrotestosterone but not by estradiol or dexamethasone. It was inhibited by R486 , a progestin antagonist, but not by flutamide, an androgen antagonist. These results indicate a mediation by the progesterone receptor. The 250-kilodalton protein appears to be an excellent probe to study in cell culture the mechanism of action of progestin on human cells.     

Rapid methods for the characterization of unilamellar phospholipid vesicles. Application to studies on fatty acid donor and acceptor properties of membranes and fatty acid binding proteins

Vesicles having diameters from 20 to 200 nm were prepared from egg-yolk phosphatidylcholine (PC) and were separated as well as analyzed by methods that can be carried out with standard laboratory equipment. Gel-chromatography on Sephacryl S 1000 was adapted for expeditious size analysis of vesicles as well as for isolation of vesicle populations having a narrow range of diameters. The internal volume of vesicles was derived from enzymic tests for PC and for glucose encapsulated. Size analysis and enzymic determinations provided a convenient check for the lamellarity of membranes produced.Fatty acids and fatty acid binding proteins (FABPs) must interact in vivo in the presence of cellular membranes. As a model, interactions between unilamellar vesicles, anthroyloxypalmitic acid (A16:0) and FABPs were studied with the aid of gel-chromatographic methods elaborated and of fluorescence spectroscopy. FABP from bovine heart donated A16:0 to membranes, whereas FABP from bovine liver removed this fatty acid from vesicle membranes. The results revealed characteristic differences between cardiac and hepatic FABPs with regard to binding a fatty acid.     

Inhibition of parathormone-stimulated bone resorption by type I interferon

The effect of Type I interferon on bone resorption was studied by measuring its effect on parathormone-stimulated calcium release from neonatal murine calvaria in vitro. A pure human recombinant leukocyte interferon hybrid of the A and D subtypes was used, which has high antiviral activity on mouse cells. Calcium release was inhibited in a dose dependent fashion with 50% inhibition at about 10(-10) M or 600 U/ml, and the inhibition was reversible. The presence of interferon was required before or during the activation phase of the resorptive response, when the formation of osteoclasts from precursor cells would occur. When added to actively resorbing bone it had no effect. The data suggest that Type I interferon can inhibit the parathormone-regulated development of active osteoclasts, possibly by inhibiting osteoclast precursor differentiation.     

Aromatization of androgens by human breast cancer.

The metabolism of dehydroepiandrosterone and testosterone by human mammary tumor was investigated. Estrogen synthesis from dehydroepiandrosterone was observed in 9 of 10 estrogen-receptor-negative tumors and only in 2 of 8 receptor-positive tumors (p less than 0.025). Conversion of testosterone to estrogens was observed in 7 of 8 receptor-negative and 2 of 7 receptor-positive tumors. Tumors which are capable of transforming dehydroepiandrosterone to estrogens were also able to aromatize testosterone suggesting that the presence of the aromatase enzyme is inherent to certain tumor cells. No estrogen formation was detected by the mitochondrial-microsomal fraction of normal breast cells while fractions from both fat cell and tumor cell showed estrogen synthesis. Estrogen formation by tumor cell fraction ranged from 5 to 190 times that observed for fat cells. The physiological significance of these results in the neoplastic tissue and its relationship to hormone dependence are discussed.     

Stimulation of the adenylate cyclase of A B16 melanoma cell line by pro-opiocortin-related peptides--a structure-activity study

The ability of alpha-melanotrophin (alpha-MSH or ACTH 1-acetyl-13 amide) and other structurally related peptides derived from the common precursor, pro-opiocortin, to stimulate adenylate cyclase activity in a pigmented B16 mouse melanoma was investigated. The peptides ACTH 1-39, ACTH 1-24, alpha-MSH, ACTH 1-13 amide and beta-MSH all stimulated the enzyme to a similar maximal extent and with similar potency (ED50 = 1.3 . 10(-6) M) except that ACTH 1-39 was slightly less potent (ED50 = 5 . 10(-6) M). ACTH 4-10 (ED50 = 4 . 10(-5) M) and gamma-MSH (ED50 = 5 . 10(-6) M) were partial agonists. ACTH 1-10 was no more effective than ACTH 4-10 in stimulating the enzyme whereas ACTH 1-13 amide was a full agonist. The peptides beta-endorphin and its derivatives, Met-enkephalin and melanotrophin potentiating factor (MPF), failed to stimulate the enzyme. We suggest that the B16 melanoma requires not only the sequence ACTH 4-10 but also some part of the sequence ACTH 11-13, or a similar sequence in the terminal portion of beta-MSH, for full activation of the receptor-linked enzyme.     

Characteristics of androgen binding in rat adrenal tissue using [3H]methyltrienolone (R1881) as tracer

A high level of binding of [3H]methyltrienolone (R1881 = 17beta-hydroxy-17alpha-methyl-estra-4, 9, 11-trien-3-one) was found in cytosol prepared from adrenals of castrated male rats. Binding of [3H]R1881 was of high affinity (DK = 6.2 nM) and highly specific for androgens. The [3H]R1881 complex migrates at 7-9S on sucrose gradients in low ionic strength buffer and at 4-5S in buffer containing 0.4M KC1. All binding studies have been performed in parallel with rat ventral prostate and adrenal cytosol. The present data suggest the presence of an androgen binding component in rat adrenal tissue.     

Ribosomal RNA homologies and the evolution of the filamentous blue-green bacteria

Summary Ribosomal RNA (rRNA) sequence homology (as determined by comparisons of T1 oligonucleotide catalogs of³²P-labeled 16S rRNAs) has been used to assess phylogenetic relationships within the filamentous and unicellular blue-green bacteria, and to identify regions of evolutionary conservatism within blue-green bacterial 16S rRNAs.Nostoc andFischerella, representatives of two morphologically distinct and highly differentiated orders, are shown to be as closely related (on the basis of RNA sequence homology) as typical members of the non-blue-green bacterial genusBacillus. They are further shown to be (on the same basis) indistinguishable from typical unicellular members of a subgroup of the unicellular blue-green bacterial order Chroococcales. These results have general implications for studies of the origin of differentiated prokaryotes and of evolutionary change in prokaryotic macromolecules. In particular, they provide indirect evidence that the divergences of contemporary major prokaryotic groups are truly ancient ones.     

Improved synthesis of 16alpha-hydroxylated androgens: intermediates of estriol formation in pregnancy.

16alpha-Hydroxyandrostenedione (16alpha-hydroxyandrost-4-ene-3,17-dione), 16alpha-hydroxytestosterone (16alpha,17beta-dihydroxyandrost-4-en-3-one) and 16alpha-hydroxydehydroepiandrosterone 3-sulfate (3beta, 16alpha-dihydroxyandrost-5-en-17-one 3-monosulfate) were synthesized by a new chemical approach with much improved yield. 16alpha-Bromoandrostendione was converted to the hydrazone of 16alpha-hydroxyandrostenedione which gave 16alpha-hydroxyandrostenedione on acid hydrolysis in total 63% yield. Oxidation of 16alpha-hydroxydehydroepiandrosterone with Jones' reagent also selectively afforded 16alpha-hydroxyandrostenedione. 16alpha-Hydroxytestosterone was observed by selective reduction of 16alpha-hydroxyandrostenedione with sodium borohydride. Reaction of 16alpha-hydroxydehydroepiandrosterone with chlorosulfonic acid in pyridine selectively gave the 3-monosulfate. The structure of the sulfate was deduced from its solvolysis to the starting material, and its acetylation and subsequent solvolysis to 16alpha-hydroxydehydroepiandrosterone 16-acetate. All procedures are suitable for large scale synthesis without the use of microorganisms.