Estimating population size and transmission bottlenecks in maternally transmitted endosymbiotic bacteria

Many species of bacterial endosymbionts are acquired by animal hosts before birth, through direct transmission from mothers to eggs or embryos. This vertical transmission imposes a reduction in numbers or "bottleneck," and the size of this bottleneck affects the population structure and evolution of symbiotic lineages. We have estimated the size of the transmission bottleneck in Buchnera, the bacterial symbiont of aphids, using basic light and electron microscopy techniques. By serial-sectioning whole aphid abdomens, their eggs, and embryos, we determined the following parameters: (i) The average size of a Buchnera cell is 2.9 mm in diameter. (ii) The total number of Buchnera in an Acyrthosiphon pisum embryo was around 36,700 whereas a first instar nymph contained more than 119,000. (iii) The number of symbionts per bacteriocyte was around 800 in an embryo and 3200 in a first instar nymph. (iv) The total number of Buchnera transmitted to each sexual egg ranged from 850 in Nasonovia to 1800 in A. pisum to more than 8000 in Uroleucon ambrosiae. (v) The total number of secondary endosymbionts in A. pisum was 12,170 for an embryo and 18,360 for a first instar nymph. Secondary symbionts were arranged both extracellularly and in clusters of 2000–8000 bacteria inside bacteriocytes. These numbers are consistent with the few previous estimates of symbiont population sizes based on counts of gene copies.     

Microplate assay measurement of cytochrome p450-carbon monoxide complexes

Cytochrome P450 in microsomes can be quantitated using the characteristic 450 nm absorption peak of the CO adduct of reduced cytochrome P450. We developed a simple microplate assay method that is superior to previous methods. Our method is less laborious, suitable for analyzing many samples, and less sensitive to sample aggregation. Microsome samples in microplate wells were incubated in a CO chamber rather than bubbled with CO gas, and then reduced with sodium hydrosulfite solution. This modification allowed a reliable and reproducible assay by effectively eliminating variations between estimations.     

Synthesis,structural studies,and biological evaluation of some purine substituted 1-aminocyclopropane-1-carboxylic acids and 1-amino-1-hydroxymethylcyclopropanes

The novel purine derivatives of 1-aminocyclopropane-1-carboxylic acid (8 and 9) and 1-amino-1-hydroxymethylcyclopropane (12 and 13) with methylene spacer between the base and the cyclopropane ring were prepared by multistep synthetic route involving alkylation of adenine and 6-(N-pyrrolyl)purine with 2-hydroxy-methyl-1-aminocyclopropane-1-carboxylic acid derivative 3 as a key reaction. All novel compounds were racemic. The N-9 substitution of the purine ring and the Z-configuration of the cyclopropane ring in 4-13 were deduced from their 1H and 13C NMR spectra by analyses of chemical shifts, H-H coupling constants and connectivities in two-dimensional homo- and heteronuclear correlation spectra. An unequivocal proof of the stereostructure of 1, 4 and 5 was obtained by their X-ray structure analysis. The novel compounds were evaluated on cytostatic and antiviral activities in several cell lines. The 6-(N-pyrrolyl)purine derivative of 1,2-aminocyclopropane alcohol 12 exhibited a more pronounced inhibitory activity against the proliferation of cervical carcinoma (HeLa) and human fibroblast (WI-38) cells than other types of tumor cell lines. None of the compounds showed inhibitory activities against cytomegalovirus, varicella-zoster virus or other viruses.     

Preparation and tumor cell uptake of poly(N-isopropylacrylamide) folate conjugates

Folate conjugates (PNIPAM-NH-FA) of a copolymer of N-isopropylacrylamide (NIPAM) and amino-N'-ethylenedioxy-bis(ethylacrylamide) were prepared by an efficient synthesis leading to random grafting, via a short dioxyethylene spacer, of approximately 7 folic acid residues per macromolecule. The chemical composition of the copolymer was characterized by (1)H NMR and UV/vis spectroscopy. A fluorophore-labeled folate PNIPAM conjugate was tested by in vitro assays performed with cultured KB-31 cells overexpressing the folate receptor. The cellular uptake of the copolymer was found to be temperature dependent and was competitively decreased by free folic acid, indicating that the polymer uptake is mediated specifically by the folate receptor. Hydrophobically modified folate conjugates of NIPAM, amino-N'-ethylenedioxy-bis(ethylacrylamide) copolymers, bearing a small number of n-octadecyl groups were prepared following a modified synthetic procedure for use in future studies of FA-targeted liposomes.     

Synthesis of arabinitol 1-phosphate and its use for characterization of arabinitol-phosphate dehydrogenase

D-arabinitol 1-phosphate (Ara-ol1-P), a substrate for D-arabinitol-phosphate dehydrogenase (APDH), was chemically synthesized from D-arabinonic acid in five steps (O-acetylation, chlorination, reduction, phosphorylation, and de-O-acetylation). Ara-ol1-P was used as a substrate for the characterization of APDH from Bacillus halodurans. APDH converts Ara-ol1-P to xylulose 5-phosphate in the oxidative reaction; both NAD(+) and NADP(+) were accepted as co-factors. Kinetic parameters for the oxidative and reductive reactions are consistent with a ternary complex mechanism.     

Plasma quercetin metabolites: structure-antioxidant activity relationships

We studied quercetin metabolism in rats to determine the nature and conjugation positions on the resulting metabolites and to evaluate their contribution to the antioxidant activity of plasma. HPLC analysis showed that quercetin is primarily metabolized to glucuronides and sulfoglucuronides and, to a minor extent, to sulfates. ESI-MS/MS studies confirmed these results and indicate that the most plausible positions for glucuronidation and sulfation are the hydroxyl groups located at positions 5 and 7, excluding the 3'-OH and 4'-OH groups. Plasma antioxidant status was significantly higher in animals to which quercetin was administrated, suggesting that quercetin metabolites can retain some antioxidant activity when the o-catechol group does not undergo conjugation reactions. It was also shown that plasma quercetin metabolites could compete in vivo with other molecules for peroxynitrite. These results enabled the establishment of quercetin metabolite structure-antioxidant activity relationships and, hence, to understand their contribution for the antioxidant potential of plasma.     

Expression of streptavidin in tomato resulted in abnormal plant development that could be restored by biotin application

Biotin is an essential cofactor for a variety of carboxylase and decarboxylase reactions and is involved in diverse metabolic pathways of all organisms. In the present study we tested the hypothesis that controlling biotin availability by the expression of Streptomyces avidinii streptavidin, would impede plant development. Transient expression of streptavidin fused to plant signal peptide, bacterial signal peptide or both, in tomato (Lycopersicon esculentum cv. VF36) plants resulted in various levels of tissue impairment, exhibited as lesion development on 1-week-old tomato seedlings. The least toxic construct was introduced to tomato (stable transformation) under the constitutive CaMV 35S promoter, and lesions appeared on stems, flower morphologies were modified and numbers and sizes of fruits were altered. Furthermore, tissue-specific expression of the streptavidin, by means of the beta-phaseolin or TobRB7 promoters, resulted in localised effects, i.e., impaired seed formation or seedless fruits, respectively, with no alteration in the morphology of the other plant organs. External application of biotin on streptavidin-expressing tomato plants prevented the degeneration symptoms and facilitated normal plant development. It can be concluded that expression of streptavidin in the plant cell can lead to local and temporal deficiencies in biotin availability, impairing developmental processes while biotin application restores plant growth cycle.