Expression of fluorescently tagged connexins: a novel approach to rescue function of oligomeric DsRed-tagged proteins

A novel, brilliantly red fluorescent protein, DsRed has become available recently opening up a wide variety of experimental opportunities for double labeling and fluorescence resonance electron transfer experiments in combination with green fluorescent protein (GFP). Unlike in the case of GFP, proteins tagged with DsRed were often found to aggregate within the cell. Here we report a simple method that allows rescuing the function of an oligomeric protein tagged with DsRed. We demonstrate the feasibility of this approach on the subunit proteins of an oligomeric membrane channel, gap junction connexins. Additionally, DsRed fluorescence was easily detected 12-16 h post transfection, much earlier than previously reported, and could readily be differentiated from co-expressed GFP. Thus, this approach can eliminate the major drawbacks of this highly attractive autofluorescent protein.     

Genetic variation of Citrus tristeza virus isolates from California and Spain: evidence for mixed infections and recombination

We examined the population structure and genetic variation of four genomic regions within and between 30 Citrus tristeza virus (CTV) isolates from Spain and California. Our analyses showed that most isolates contained a population of sequence variants, with one being predominant. Four isolates showed two major sequence variants in some genomic regions. The two major variants of three of these isolates showed very low nucleotide identity to each other but were very similar to those of other isolates, suggesting the possibility of mixed infections with two divergent isolates. Incongruencies of phylogenetic relationships in the different genomic regions and statistical analyses suggested that the genomes of some CTV sequence variants originated by recombination events between diverged sequence variants. No correlation was observed between geographic origin and nucleotide distance, and thus from a genetic view, the Spanish and Californian isolates analyzed here could be considered members of the same population.     

M-DNA: A complex between divalent metal ions and DNA which behaves as a molecular wire

M-DNA is a complex of DNA with divalent metal ions (Zn(2+), Co(2+), or Ni(2+)) which forms at pH conditions above 8. Upon addition of these metal ions to B-DNA at pH 8.5, the pH decreases such that one proton is released per base-pair per metal ion. Together with previous NMR data, this result demonstrated that the imino proton in each base-pair of the duplex was substituted by a metal ion and that M-DNA might possess unusual conductive properties. Duplexes of 20 base-pairs were constructed with fluorescein (donor) at one end and rhodamine (acceptor) at the other. Upon formation of M-DNA (with Zn(2+)) the fluorescence of the donor was 95 % quenched. Fluorescence lifetime measurements showed the presence of a very fast component in the decay kinetics with tau相似文献   

The morphological transition of Helicobacter pylori cells from spiral to coccoid is preceded by a substantial modification of the cell wall.

The peptidoglycan (murein) of Helicobacter pylori has been investigated by high-performance liquid chromatography and mass spectrometric techniques. Murein from H. pylori corresponded to the A1gamma chemotype, but the muropeptide elution patterns were substantially different from the one for Escherichia coli in that the former produced high proportions of muropeptides with a pentapeptide side chain (about 60 mol%), with Gly residues as the C-terminal amino acid (5 to 10 mol%), and with (1-->6)anhydro-N-acetylmuramic acid (13 to 18 mol%). H. pylori murein also lacks murein-bound lipoprotein, trimeric muropeptides, and (L-D) cross-linked muropeptides. Cessation of growth and transition to coccoid shape triggered an increase in N-acetylglucosaminyl-N-acetylmuramyl-L-Ala-D-Glu (approximately 20 mol%), apparently at the expense of monomeric muropeptides with tri- and tetrapeptide side chains. Muropeptides with (1-->6)anhydro-muramic acid and with Gly were also more abundant in resting cells.     

A quantitative and qualitative reanalysis of the endocast from the juvenile Paranthropus specimen l338y-6 from Omo, Ethiopia

Based on an analysis of its endocast, Holloway (1981 Am J Phys Anthropol 53:109-118) attributed the juvenile Omo L338y-6 specimen to Australopithecus africanus (i.e., gracile australopithecines) rather than to Paranthropus (Australopithecus) boisei (robust australopithecines) favored by other workers (Rak and Howell [1978] Am J Phys Anthropol 48:345-366). Holloway's attribution was based on the specimen's (1) low cranial capacity, (2) gracile-like meningeal vessels, (3) gracile-like cerebellar hemispheres, and (4) absence of an enlarged occipital/marginal (O/M) sinus system. Recent work, however, has shown that criteria 1 and 2 are not useful for sorting gracile from robust australopithecines (Culotta [1999] Science 284:1109-1111; Falk [1993] Am J Phys Anthropol 92:81-98). In this paper, we test criterion 3 by quantifying the endocranial cerebellar and occipital morphology reproduced on the Omo L338y-6 endocast, and comparing it to seven endocasts from South and East African early hominids. Our preliminary results show that metric analysis of this specimen cannot be used to sort it preferentially with either robust or gracile australopithecines. Finally, we demonstrate that, contrary to previous reports, the Omo L338y-6 endocast reproduces an enlarged left occipital sinus (criterion 4). This observation is consistent with the original attribution of the Omo specimen to robust australopithecines (Rak and Howell [1978] Am J Phys Anthropol 48:345-366). Furthermore, if Omo L338y-6 was a robust australopithecine, this discovery extends the occurrence of an enlarged O/M sinus system to one of the earliest known paranthropines. Am J Phys Anthropol 110:399-406, 1999.     

Tumor attenuation by 2(6-hydroxynaphthyl)-beta-D-xylopyranoside requires priming of heparan sulfate and nuclear targeting of the products

We have previously reported that the heparan sulfate-priming glycoside 2-(6-hydroxynaphthyl)-beta-D-xylopyranoside selectively inhibits growth of transformed or tumor-derived cells. To investigate the specificity of this xyloside various analogs were synthesized and tested in vitro. Selective growth inhibition was dependent on the presence of a free 6-hydroxyl in the aglycon. Because cells deficient in heparan sulfate synthesis were insensitive to the xyloside, we conclude that priming of heparan sulfate synthesis was required for growth inhibition. In growth-inhibited cells, heparan sulfate chains primed by the active xyloside were degraded to products that contained anhydromannose and appeared in the nuclei. Hence the degradation products were generated by nitric oxide-dependent cleavage. Accordingly, nitric oxide depletion reduced nuclear localization of the degradation products and counteracted the growth-inhibitory effect of the xyloside. We propose that 2-(6-hydroxynaphthyl)-beta-D-xylopyranoside entered cells and primed synthesis of heparan sulfate chains that were subsequently degraded by nitric oxide into products that accumulated in the nucleus. In vivo experiments demonstrated that the xyloside administered subcutaneously, perorally, or intraperitoneally was adsorbed and made available to tumor cells located subcutaneously. Treatment with the xyloside reduced the average tumor load by 70-97% in SCID mice. The present xyloside may serve as a lead compound for the development of novel antitumor strategies.     

Sequence Analysis of DNA Fragments from the Genome of the Primary Endosymbiont of the Whitefly <Emphasis Type="Italic">Bemisia tabaci</Emphasis>

The whitefly Bemisia tabaci contains a primary prokaryotic endosymbiont housed within specialized cells in the body cavity. Two DNA fragments from the endosymbiont, totaling 33.3 kilobases, were cloned and sequenced. In total, 37 genes were detected and included the ribosomal RNA operon and genes for ribosomal RNA proteins. The guanine plus cytosine of the DNA was 30.2 mol%, different from that of endosymbionts of other plant sap-sucking insects.     

Thrombin inhibitor reduces myocardial infarction in apoE-/- x LDLR-/- mice

We have previously shown that atherosclerotic apolipoprotein E-deficient (apoE(-/-)) x LDL receptor-deficient (LDLR(-/-)) mice develop myocardial infarction when exposed to hypoxic stress. This study was performed to assess the role of thrombin and thrombosis in this process. ApoE(-/-) x LDLR(-/-) mice were fed a cholesterol-rich diet for 8 mo and were then subjected to hypoxic stress while receiving isoflurane anesthesia. One group received a bolus dose (5.6 micromol/kg) of the thrombin inhibitor melagatran, and control animals received PBS 10 min before the hypoxic stress. The mice were exposed to 10 min of hypoxia followed by normoxia. Ten minutes after the stress, Alzet pumps delivering melagatran (20 nmol x kg x (-1)min(-1)) or PBS were implanted, and the mice were allowed to recover for 48 h. The cardiac response was analyzed by histology, immunohistochemistry, and serum troponin T assay. All animals showed reversible ECG changes as a sign of ischemia during hypoxic stress, and 50% developed infarctions afterward as judged by troponin T levels. The group that received thrombin inhibitor had significantly lower troponin T and smaller myocardial infarctions than the PBS-treated group. These data show that thrombin generation is an important pathogenetic factor and suggest that coronary thrombosis is involved in myocardial infarction in atherosclerotic mice. Exposure of atherosclerotic mice to hypoxia leads to myocardial infarction through a two-phase pathway in which acute transient ischemia is followed by thrombin-dependent, irreversible, myocardial ischemia and myocardial cell death.