Possible origins of CTnBST, a conjugative transposon found recently in a human colonic Bacteroides strain

A previous survey of Bacteroides isolates suggested that the ermB gene entered Bacteroides spp. recently. Previously, ermB had been found almost exclusively in gram-positive bacteria. In one Bacteroides strain, ermB was located on 100-kb conjugative transposon (CTn) CTnBST. To assess the possible origin of this CTn, we obtained the full DNA sequence of CTnBST and used this information to investigate its possible origins. Over one-half of CTnBST had high sequence identity to a putative CTn found in the genome of Bacteroides fragilis YCH46. This included the ends of the CTn and genes involved in integration, transfer, and excision. However, the region around the ermB gene contained genes that appeared to originate from gram-positive organisms. In particular, a 7-kb segment containing the ermB gene was 100% identical to an ermB region found in the genome of the gram-positive bacterium Arcanobacterium pyogenes. A screen of Bacteroides isolates whose DNA cross-hybridized with a CTnBST probe revealed that several isolates did not carry the 7-kb region, implying that the acquisition of this region may be more recent than the acquisition of the entire CTnBST element by Bacteroides spp. We have also identified other Bacteroides isolates that carry a slightly modified 7-kb region but have no other traces of CTnBST. Thus, it is possible that this 7-kb region could itself be part of a mobile element that has inserted in a Bacteroides CTn. Our results show that CTnBST is a hybrid element which has acquired a portion of its coding region from gram-positive bacteria but which may originally have come from Bacteroides spp. or some related species.     

Blue light induces radical formation and autophosphorylation in the light-sensitive domain of Chlamydomonas cryptochrome

Cryptochromes are sensory blue light receptors mediating various responses in plants and animals. Studies on the mechanism of plant cryptochromes have been focused on the flowering plant Arabidopsis. In the genome of the unicellular green alga Chlamydomonas reinhardtii, a single plant cryptochrome, Chlamydomonas photolyase homologue 1 (CPH1), has been identified. The N-terminal 500 amino acids comprise the light-sensitive domain of CPH1 linked to a C-terminal extension of similar size. We have expressed the light-sensitive domain heterologously in Escherichia coli in high yield and purity. The 59-kDa protein bears exclusively flavin adenine dinucleotide in its oxidized state. Illumination with blue light induces formation of a neutral flavin radical with absorption maxima at 540 and 580 nm. The reaction proceeds aerobically even in the absence of an exogenous electron donor, which suggests that it reflects a physiological response. The process is completely reversible in the dark and exhibits a decay time constant of 200 s in the presence of oxygen. Binding of ATP strongly stabilizes the radical state after illumination and impedes the dark recovery. Thus, ATP binding has functional significance for plant cryptochromes and does not merely result from structural homology to DNA photolyase. The light-sensitive domain responds to illumination by an increase in phosphorylation. The autophosphorylation takes place although the protein is lacking its native C-terminal extension. This finding indicates that the extension is dispensable for autophosphorylation, despite the role it has been assigned in mediating signal transduction in Arabidopsis.     

Water permeability differs between growing and non-growing barley leaf tissues

A pressure probe technique and an osmotic swelling assay were used to compare water transport properties between growing and non-growing tissues of leaf three of barley. The epidermis was analysed in planta by pressure probe, whereas (predominantly) mesophyll protoplasts were analysed by osmotic swelling. Hydraulic conductivity (Lp) and, by implication, water permeability (Pf) of epidermal cells was 31% higher in the leaf elongation zone (Lp=0.5+/-0.2 microm s-1 MPa-1; Pf=65+/-25 microm s-1; means+/-SD of n=17 cells) than in the, non-growing, emerged leaf zone (Lp=0.4+/-0.1 microm s-1 MPa-1; Pf=50+/-15 microm s-1; n=24; P<0.05). Similarly, water permeability of mesophyll protoplasts was by 55% higher in the elongation compared with emerged leaf zone (Pf=13+/-1 microm s-1 compared with 8+/-1 microm s-1; n=57 and 36 protoplasts, respectively; P<0.01). Within the leaf elongation zone, a small population of larger-sized protoplasts could be distinguished. These protoplasts, which originated most likely from parenchymateous bundle sheath or midrib parenchyma cells, had a three-fold higher water permeability (P<0.001) as mesophyll protoplasts. The effect on Lp and Pf of known aquaporin inhibitors was tested with the pressure probe (Au+, Ag+, Hg2+, phloretin) and the osmotic swelling assay (phloretin). Only phloretin, when applied to protoplasts in the swelling assay caused an average decrease in Pf, but the effect varied between isolations. Technical approaches and cell-type and growth-specific differences in water transport properties are discussed.     

Leptin restores plasma cholesterol, glucose and weight loss induced by IFNalpha treatment

Leptin, an adipokine, a major regulator of food intake, was recently suggested to play a role in immune response. We previously showed that weight reduction following IFNalpha therapy is due, at least in part, to direct induction of adipose tissue apoptosis. We now studied the effect of leptin on IFNalpha treated adipocytes in vitro and in vivo. Diet induced obese C57/B6 mice were treated continually with recombinant (r) IFNalphaA/D + leptin (100 U/g body weight + 10 microg/day, respectably) or leptin (10 microg/day) alone for 8 days. Co-administration of IFNalphaA/D + leptin significantly reduced plasma cholesterol (P<0.001), glucose (P<0.007) and pro-apoptotic protein levels (P<0.05). Additionally, co-administration prevented loss of body weight due to adipocyte apoptosis. Thus, leptin co-administration with IFNalphaA/D decreases some of the side effects of IFNalpha administration such as weight loss, cholesterol and glucose levels.     

The purification and characterization of alpha interferons and related cytokine receptors--a personal account

In 1976–1977, I adapted reversed-phase HPLC (RP-HPLC) to peptide and protein purification, starting with pituitary proteins and continuing with the first successful purification to homogeneity of human leukocyte interferon (IFN-). Using this technology, I isolated and characterized 6–8 different leukocyte interferon subtypes, which were later identified as products of the IFN- gene family. Since then, RP-HPLC became a standard procedure for isolation and analysis of proteins. The successful purification of IFN- led to the development of Roferon-A™, a drug used for the treatment of hairy cell leukemia, hepatitis C and a variety of other diseases. Later studies with my colleagues in Israel and abroad led to isolation and discovery of several cytokine receptors and binding proteins, including those of Type I IFNs, TNF and IL-18. The use of HPLC was indispensable in most of these studies.     

Formation of polysaccharide gel layers in the presence of Ca2+ and K+ ions: measurements and mechanisms

Understanding the adsorption properties of polysaccharides in terms of substrate affinity, kinetics, and layer structure is of paramount importance in numerous industrial and natural systems. The structural growth of the layers of two model polysaccharides--sodium alginate and polygalacturonic acid (PGA)--was characterized by quartz crystal microbalance with dissipation and atomic force microscopy. Monitoring the variations in frequency and dissipation energy provides insights into both the average adsorbed mass and the viscoelastic properties of the adsorbed layer of polyelectrolytes along with the associated ions and water molecules. Both polysaccharides had similar adsorption patterns with increasing ionic strengths and showed significant complexation of calcium ions. In the presence of calcium, the alginate gel layer exhibited significant swelling with an increasing concentration of monovalent salt that the PGA gel layer did not manifest. Basing our discussion on the "egg-box model", we interpreted these different swelling behaviors as resulting from differences in the complexation modes of the two polysaccharides. The dimerization of the polymers by cross-linking and the weaker dimer-dimer associations play major roles in the sensitivity of the polysaccharide gel matrix to high salt concentration environments.