Stereoselective biosynthesis of chloroarylpropane diols by the basidiomycete Bjerkandera adusta: exploring the roles of amino acids, pyruvate, glycerol and phenyl acetyl carbinol

Bjerkandera adusta produces many chlorometabolites including chlorinated anisyl metabolites (CAMs) and 1-arylpropane-1,2-diols (1, 2, 3, 4) as idiophasic metabolic products of L-phenylalanine. These diols are stereoselectively biosynthesized from a C7-unit (benzylic, from L-phenylalanine) and a C2-unit, of unknown origin, as predominantly erythro (1R,2S) enantiomers. Of the labeled amino acids tested as possible C2-units, at the 4-10 mM level, none were found to efficiently label the 2,3-propane carbons of the diols. However, glycine (2-13C), L-serine (2,3,3-d3) and L-methionine (methyl-d3) entered the biomethylation pathway. Neither pyruvate (2,3-13C2), acetate (1,2-13C2), acetaldehyde (d4) nor ethanol (ethyl-d5) labeled the 2,3-propane carbons of the diols at the 4-10 mM level. Pyruvate (2,3-13C2) and L-serine (2,3,3-d3) (which also entered the biomethylation pathway) did, however, effectively label the 2,3-propane carbons of the alpha-ketols and diols at the 40 mM level as evidenced by mass spectrometry. Glycerol (1,1,2,3,3-d5) also appeared to label one of the 2,3-propane carbons (ca. 5% as 2H2 in the C3 side chain) as suggested by mass spectrometric data and also entered the biomethylation pathway, likely via amino acid synthesis. Glycerol (through pyruvate), therefore, likely supplies C2 and C3 of the propane side chain with arylpropane diol biosynthesis. Incubation of B. adusta with synthetic [2-2H1, 2-18O]-glycerol showed that neither 2H nor 18O were incorporated in the alpha-ketols or diols. The oxygen atom on the C2 of the ketols/diols, therefore, does not appear to come from the oxygen atom on the C2 of glycerol. Glycerol, however, can readily form L-serine (which can then form pyruvate via PLP/serine dehydratase and involve transamination washing out the 18O label and providing the oxygen from water), and can then go on to label the C2-unit. Labeled alpha-ketol, phenyl acetyl carbinol (5) (PAC; ring-d(5), 2,3-13C2 propane) cultured with B. adusta leads to stereospecific reduction to the (1R,2S)-diol (6) (ring-d5 and 2,3-13C2); in all other metabolites produced, the 2,3-13C2) label is washed out. Incubation of the fungus with 4-fluorobenzaldehyde (13) produces a pooling of predominantly erythro (1R,2S) 1-(4'-fluorophenyl)-1,2-propane diol (18 as diacetate) (through the corresponding alpha-ketols 16, 17). Blocking the para-position with fluorine thus appears to prevent ring oxygenation and also chlorination, forcing the conclusion that para-ring oxygenation precedes meta-chlorination.     

Dynamic rearrangement of the filamentous actin network occurs during zoosporogenesis and encystment in the oomycete phytophthora cinnamomi

The organization of filamentous actin (F-actin) in living cells of the oomycete Phytophthora cinnamomi was determined during zoosporogenesis and zoospore encystment by microinjecting sporangia with fluorescently labeled phalloidin and observing resultant fluorescence by confocal microscopy. In multinucleate sporangia prior to the induction of cleavage, phalloidin labeling took the form of plaques which occurred mainly in the periphery of the sporangia. After induction of cleavage, phalloidin labeling showed that the plaques disappeared and that F-actin began to accumulate along the developing cleavage planes and around nuclei and water expulsion vacuoles. F-actin labeling was also observed near the plasma membrane in zoospores and young cysts but reverted to the plaque form in older cysts. Localization of F-actin close to the developing cleavage planes is consistent with the idea that actin microfilaments function in the positioning and expansion of the cleavage membranes. Observations of plaques of actin in living sporangia provide evidence that plaques are not aldehyde-induced fixation artifacts. Copyright 1998 Academic Press.     

Serglycin expression during monocytic differentiation of U937-1 cells

Serglycin is the major proteoglycan in most hematopoietic cells, including monocytes and macrophages. The monoblastic cell line U937-1 was used to study the expression of serglycin during proliferation and differentiation. In unstimulated proliferating U937-1 cells serglycin mRNA is nonconstitutively expressed. The level of serglycin mRNA was found to correlate with the synthesis of chondroitin sulfate proteoglycan (CSPG). The U937-1 cells were induced to differentiate into different types of macrophage-like cells by exposing the cells to PMA, RA, or VitD3. These inducers of differentiation affected the expression of serglycin mRNA in three different ways. The initial upregulation seen in the normally proliferating cells was not observed in PMA treated cells. In contrast, RA increased the initial upregulation, giving a reproducible six times increase in serglycin mRNA level from 4 to 24 h of incubation, compared to a four times increase in the control cells. VitD3 had no effect on the expression of serglycin mRNA. The incorporation of (35S)sulfate into CSPG decreased approximately 50% in all three differentiated cell types. Further, the (35S)CSPGs expressed were of larger size in PMA treated cells than controls, but smaller after RA treatment. This was due to the expression of CSPGs, with CS-chains of 25 and 5 kDa in PMA and RA treated cells, respectively, compared to 11 kDa in the controls. VitD3 had no significant effect on the size of CSPG produced. PMA treated cells secreted 75% of the (35S)PGs expressed, but the major portion was retained in cells treated with VitD3 or RA. The differences seen in serglycin mRNA levels, the macromolecular properties of serglycin and in the PG secretion patterns, suggest that serglycin may have different functions in different types of macrophages.      

Human placenta contains a high affinity R1881 binding site that is not the androgen receptor

Human placental cytosol was shown to contain a species that binds the synthetic androgen, methyltrienolone (R1881) with high affinity (Kd 6.5 nM). Major differences were found between this placental androgen binding species and the classical androgen receptor found in human foreskin cytosol. Competitive binding assays in the placental cytosol using [3H]R1881 as tracer showed a 200-fold excess of testosterone to compete poorly, while dihydrotestosterone and the synthetic androgen mibolerone did not compete at all. The placental R1881 binding component was found not to bind to hydroxylapatite, although all classes of steroid receptors are reported to do so. Temperature studies showed that the placental binding site is stable at elevated temperatures with no loss of binding after 4 h at 45 degrees C. Ion exchange chromatography showed that the placental R1881 binding site eluted from DEAE cellulose at a lower salt concentration than foreskin androgen receptors. These results show that R1881 is not entirely specific for androgen receptors and that human placenta contains an androgen binding site that is not the classical androgen receptor.     

Acute IL-6 treatment increases fatty acid turnover in elderly humans in vivo and in tissue culture in vitro

To determine whether IL-6 increases lipolysis and fat oxidation in patients with type 2 diabetes and/or whether it exerts this effect independently of changes to the hormonal milieu, patients with type 2 diabetes (D) and healthy control subjects (CON) underwent recombinant human (rh)IL-6 infusion for 3 h. Rates of appearance (Ra) and disappearance (Rd) of [U-(13C)]palmitate and [6,6-(2H2)]glucose were determined. rhIL-6 infusion increased (P < 0.05) palmitate Ra and Rd in a similar fashion in both groups. Neither plasma glucose concentration nor glucose Ra/Rd was affected by rhIL-6 infusion in either group, whereas rhIL-6 infusion resulted in a reduction (P < 0.05) in circulating insulin in D. Plasma growth hormone (GH) was increased (P < 0.05) by IL-6 in CON, and cortisol increased (P < 0.05) in response to IL-6 in both groups. To determine whether IL-6 was exerting its effect directly or through activation of these hormones, we performed cell culture experiments. Fully differentiated 3T3-L1 adipocytes were treated with PBS (control) IL-6, or IL-6 plus dexamethasone and GH. IL-6 treatment alone increased (P < 0.05) lipolysis, but this effect was reduced by the addition of dexamethasone and GH such that IL-6 plus dexamethasone and GH had blunted (P < 0.05) lipolysis compared with IL-6 alone. To assess whether IL-6 increases fat oxidation, L6 myotubes were treated with PBS (Control), IL-6, or AICAR, a compound known to increase lipid oxidation. Both IL-6 and AICAR markedly increased (P < 0.05) oxidation of [(14)C]palmitate compared with Control. Acute IL-6 treatment increased fatty acid turnover in D patients as well as healthy CON subjects. Moreover, IL-6 appears to be activating lipolysis independently of elevations in GH and/or cortisol and appears to be a potent catalyst for fat oxidation in muscle cells.     

Do the four clades of the mtDNA haplogroup L2 evolve at different rates?

Forty-seven mtDNAs collected in the Dominican Republic and belonging to the African-specific haplogroup L2 were studied by high-resolution RFLP and control-region sequence analyses. Four sets of diagnostic markers that subdivide L2 into four clades (L2a-L2d) were identified, and a survey of published African data sets appears to indicate that these clades encompass all L2 mtDNAs and harbor very different geographic/ethnic distributions. One mtDNA from each of the four clades was completely sequenced by means of a new sequencing protocol that minimizes time and expense. The phylogeny of the L2 complete sequences showed that the two mtDNAs from L2b and L2d seem disproportionately derived, compared with those from L2a and L2c. This result is not consistent with a simple model of neutral evolution with a uniform molecular clock. The pattern of nonsynonymous versus synonymous substitutions hints at a role for selection in the evolution of human mtDNA. Regardless of whether selection is shaping the evolution of modern human mtDNAs, the population screening of L2 mtDNAs for the mutations identified by our complete sequence study should allow the identification of marker motifs of younger age with more restricted geographic distributions, thus providing new clues about African prehistory and the origin and relationships of African ethnic groups.     

Tracing European founder lineages in the Near Eastern mtDNA pool

Founder analysis is a method for analysis of nonrecombining DNA sequence data, with the aim of identification and dating of migrations into new territory. The method picks out founder sequence types in potential source populations and dates lineage clusters deriving from them in the settlement zone of interest. Here, using mtDNA, we apply the approach to the colonization of Europe, to estimate the proportion of modern lineages whose ancestors arrived during each major phase of settlement. To estimate the Palaeolithic and Neolithic contributions to European mtDNA diversity more accurately than was previously achievable, we have now extended the Near Eastern, European, and northern-Caucasus databases to 1,234, 2, 804, and 208 samples, respectively. Both back-migration into the source population and recurrent mutation in the source and derived populations represent major obstacles to this approach. We have developed phylogenetic criteria to take account of both these factors, and we suggest a way to account for multiple dispersals of common sequence types. We conclude that (i) there has been substantial back-migration into the Near East, (ii) the majority of extant mtDNA lineages entered Europe in several waves during the Upper Palaeolithic, (iii) there was a founder effect or bottleneck associated with the Last Glacial Maximum, 20,000 years ago, from which derives the largest fraction of surviving lineages, and (iv) the immigrant Neolithic component is likely to comprise less than one-quarter of the mtDNA pool of modern Europeans.