Fluorescence resonance energy transfer analysis of the structure of the four-way DNA junction.

We have carried out fluorescence resonance energy transfer (FRET) measurements on four-way DNA junctions in order to analyze the global structure and its dependence on the concentration of several types of ions. A knowledge of the structure and its sensitivity to the solution environment is important for a full understanding of recombination events in DNA. The stereochemical arrangement of the four DNA helices that make up the four-way junction was established by a global comparison of the efficiency of FRET between donor and acceptor molecules attached pairwise in all possible permutations to the 5' termini of the duplex arms of the four-way structure. The conclusions are based upon a comparison between a series of many identical DNA molecules which have been labeled on different positions, rather than a determination of a few absolute distances. Details of the FRET analysis are presented; features of the analysis with particular relevance to DNA structures are emphasized. Three methods were employed to determine the efficiency of FRET: (1) enhancement of the acceptor fluorescence, (2) decrease of the donor quantum yield, and (3) shortening of the donor fluorescence lifetime. The FRET results indicate that the arms of the four-way junction are arranged in an antiparallel stacked X-structure when salt is added to the solution. The ion-related conformational change upon addition of salt to a solution originally at low ionic strength progresses in a continuous noncooperative manner as the ionic strength of the solution increases. The mode of ion interaction at the strand exchange site of the junction is discussed.     

Unique poly(dA).poly(dT) B'-conformation in cellular and synthetic DNAs

Poly(dA).poly(dT), but not B-form DNA, is specifically recognized by experimentally induced anti-kinetoplast or anti-poly(dA).poly(dT) immunoglobulins. Antibody binding is completely competed by poly(dA).poly(dT) and poly(dA).poly(dU) but not by other single- or double-stranded DNA sequences in a right-handed B-form. Antibody interaction with poly(dA).poly(dT) depends on immunoglobulin concentration, incubation time and temperature, and is sensitive to elevated ionic strengths. Similar conformations, for example, (dA)4-6 X (dT)4-6, in the kinetoplast DNA of the parasite Leishmania tarentolae are also immunogenic and induce specific anti-poly(dA).poly(dT) antibodies. These antibody probes specifically recognize nuclear and kinetoplast DNA in fixed flagellated kinetoplastid cells as evidenced by immunofluorescence microscopy. Anti-poly(dA).poly(dT) immunofluorescence is DNase-sensitive and competed by poly(dA).poly(dT), but not other classical double-stranded B-DNAs. Thus, these unique cellular B'-DNA helices are immunogenic and structurally similar to synthetic poly(dA).poly(dT) helices in solution.     

Decline in measles mortality: nutrition,age at infection,or exposure?

The mortality from measles was studied in an urban area of Guinea-Bissau one year before and five years after the introduction of a vaccination programme. The years after the introduction of immunisation saw a decline in mortality among unvaccinated children with measles. This decline occurred despite a lower age at infection and an increasing prevalence of malnourished children. State of nutrition (weight for age) did not affect the outcome of measles infection. The incidence of isolated cases, however, increased in the period after the introduction of measles vaccination. As mortality was lower among these cases, diminished clustering explained some of the reduction in mortality. Comparison between the urban district and a rural area inhabited by the same ethnic group showed a lower age at infection, less clustering of cases, and lower case fatality ratios in the urban area.Endemic transmission of measles in urban districts leads to less clustering of cases, which may help explain the usually lower case fatality ratios in these areas. As measles vaccination increases herd immunity and diminishes clustering of cases, it may reduce mortality even among unvaccinated children who contract the disease.     

Floral scent in dioeciousSalix (Salicaceae)—a cue determining the pollination system?

Floral scents of male and female inflorescences of three dioeciousSalix species were collected by head-space adsorption, and analysed by GC-MS. InSalix caprea andS. cinerea 1,4-dimethoxy benzene was the main compound, and male and female scents showed a high degree of resemblance. No dominant compound was found inS. repens and malefemale scent similarity was low. Floral scent inSalix is likely a strong orientation cue, guiding pollinators between male and female plants ensuring pollen transfer and pollination. We suggest that a high degree of male-female floral scent resemblance is coupled to a high degree of insect pollination. Floral scent does not promote reproductive isolation betweenS. caprea andS. cinerea.     

Iodoazomycin riboside (1-(5'-iodo-5'-deoxyribofuranosyl)-2-nitroimidazole), a hypoxic cell marker. I. Synthesis and in vitro characterization

Misonidazole (MISO), a selective radiosensitizer of hypoxic cells, forms adducts with cellular biomolecules with rates which are 30-50 X higher under hypoxic as compared to aerobic conditions of incubation. This technique of sensitizer adduct formation was proposed as a possible means of measuring the hypoxic fraction of solid tumors by noninvasive procedures. Iodoazomycin riboside (5'-IAZR) and 5'-[125I]AZR were synthesized and chemically characterized. Measurements of in vitro cytotoxicity and radiosensitizing ability with EMT-6 tumor cells in vitro indicated that 5'-IAZR is approximately 3 X more toxic and effective than is azomycin riboside (AZR) and approximately 10 X more toxic and effective than is MISO. 5'-[125I]AZR was shown to selectively bind to hypoxic EMT-6 cells at rates which were 2.5-3 X faster than those of MISO. The absolute rates of binding of 5'-IAZR to hypoxic cells at concentrations of 10-100 microM are the highest observed in this laboratory for any hypoxic cell radiosensitizer tested to date. These data suggest that 5'-IAZR, when labeled with an appropriate radioisotope (e.g., 131I), might be a useful marker for hypoxic cells in solid tumors amenable to noninvasive detection. Additional studies with animal tumor models appear to be warranted.     

Floral scent variation in the Pyrola rotundifolia complex in Scandinavia and Western Greenland

The variation in floral scent composition within and among populations of four taxa belonging to the Pyrola rotundifolia complex was analyzed using Principal Component Analysis. Two major groups were recognized, P. norvegica and P. grandiflora on the one side and P. rotundifolia s. str. on the other. Benzaldehyde dominated the scent of the first group and methoxy benzenes and phenyl propanoids that of the second group. A large variation in the floral scent chemistry was found both within some of the studied populations as well as among them. The floral scent composition of P. rotundifolia ssp. maritima was no more different from P. rotundifolia ssp. rotundifolia , than the differences between populations of P. rotundifolia ssp. rotundifolia . The findings are in partial agreement with the current delimitation of the taxa in the P. rotundifolia complex.     

Effect of environmental pH on the fermentation balance of Lactobacillus reuteri

The influence of environmental pH on the regulation of glucose catabolism by Lactobacillus reuteri was examined in anaerobic batch cultures. Under acidic conditions both glucose consumption and end-products formation were low. Maximum biomass was reached at pH 5·0, with a specific growth rate of µ= 0·78 h^-1. The shift in pH values from 4.3 to 6.5 reflected an increase in glucose uptake as well as in the yield ( Y _p/x) of acetate, lactate and ethanol after 12 h of incubation. Ethanol was the major metabolite produced at all pH values assayed.     

Rac GTPase interacts with GAPs and target proteins through multiple effector sites.

Rac, a small GTPase in the ras superfamily, regulates at least two biological processes in animal cells: (i) the polymerization of actin and the assembly of integrin complexes to produce lamellipodia and ruffles; and (ii) the activity of an NADPH oxidase in phagocytic cells. NADPH oxidase activation is mediated through a rac effector protein, p67phox, and using chimeras made between rac and the closely related GTPase, rho, we have identified two distinct effector sites in rac, one N-terminal and one C-terminal, both of which are required for activation of p67phox. The same two effector sites are essential for rac-induced actin polymerization in fibroblasts. p65PAK, a ubiquitous serine/threonine kinase, interacts with rac at both the N- and C-terminal effector sites, but the GTPase-activating protein, bcr interacts with rac at a different region. This makes p65PAK, but not bcr, a candidate effector of rac-induced lamellipodium formation.