Supercurrent in a Double Quantum Dot.

We demonstrate the Josephson effect in a serial double quantum dot defined in a nanowire with epitaxial superconducting leads. The supercurrent stability diagram adopts a honeycomb pattern. We observe sharp discontinuities in the magnitude of the critical current, I_{c}, as a function of dot occupation, related to doublet to singlet ground state transitions. Detuning of the energy levels offers a tuning knob for I_{c}, which attains a maximum at zero detuning. The consistency between experiment and theory indicates that our device is a faithful realization of the two-impurity Anderson model.

Spatial and temporal control of transgene expression in vivo using a heat-sensitive promoter and MRI-guided focused ultrasound.

BACKGROUND: Among the techniques used to induce and control gene expression, a non-invasive, physical approach based on local heat in combination with a heat-sensitive promoter represents a promising alternative but requires accurate temperature control in vivo. MRI-guided focused ultrasound (MRI-FUS) with real-time feedback control allows automatic execution of a predefined temperature-time trajectory. The purpose of this study was to demonstrate temporal and spatial control of transgene expression based on a well-defined local hyperthermia generated by MRI-FUS. METHODS: Expression of the green fluorescent protein (GFP) marker gene was used. Two cell lines were derived from C6 glioma cells. The GFP expression of the first one is under the control of the CMV promoter, whereas it is under the control of the HSP70 promoter in the second one and thus inducible by heat. Subcutaneous tumours were generated by injection in immuno-deficient mice and rats. Tumours were subjected to temperatures varying from 42 to 50 degrees C for 3 to 25 min controlled by MRI-FUS and analyzed 24 h after the heat-shock. Endogenous HSP70 expression and C6 cell distribution were also analyzed. RESULTS: The results demonstrate strong expression at 50 degrees C applied during a short time period (3 min) without affecting cell viability. Induced expression was also clearly shown for temperature in the range 44-48 degrees C but not at 42 degrees C. CONCLUSIONS: Heating with MRI-FUS allows a tight and non-invasive control of transgene expression in a tumour.

Encapsulation of deoxyribonucleic acid molecules in silica and hybrid organic-silica gels.

The encapsulation of small DNA molecules was attempted in pure silica and in hybrid polyvinyl alcohol-silica gels. The materials which were obtained were examined by nitrogen adsorption, and by 29Si and 31P NMR spectroscopy. The extraction of the DNA molecules from the gels was examined in a buffer aqueous solution as well as in an acidic medium. The results suggested that the DNA molecules remained trapped inside the gels due to a permanent bonding to the gel network.

Control of transgene expression using local hyperthermia in combination with a heat-sensitive promoter.

BACKGROUND: Local production of therapeutic proteins, e.g. for cancer treatments, is based on gene therapy approaches and requires tight spatial and temporal control of gene expression. Here we demonstrate the use of local hyperthermia of varying intensity and duration to control the expression of a transgene under control of the thermoinducible hsp70 (heat shock protein) promoter. METHODS: Heat-induced expression of the EGFP (green fluorescent protein) reporter gene was characterized using a stably transfected glioma C6 cell line expressing the EGFP gene under control of the heat inducible minimal hsp70 promoter both in vitro and in vivo for subcutaneous tumors in immunodeficient mice. RESULTS: A heat shock of 20-30 min at 43 degrees C in cell culture led to a maximum EGFP concentration at about 24 h. Heat treatments at higher temperature (up to 48 degrees C) but with shorter durations (down to 30 s) also induced strong EGFP expression. Local heating in situ led to gradients in EGFP expression which decreased with increasing distance from the heat source. CONCLUSION: Local hyperthermia, in combination with a heat sensitive promoter, represents a method for the spatial and temporal control of transgene expression.

A protein with the characters of a zinc-finger is implicated in the differentiation of Schwann cells.

During the development of the PNS, Schwann cells (SC) differentiate into myelinating and nonmyelinating cells, implying regulation by different transcription factors such as ZF proteins. Employing an original strategy using monoclonal antibodies specifically directed against the conserved ZF motif, we have identified a new ZF protein of 55 kDa present in rat sciatic nerve extract (SCp55). We used polyclonal antibodies and cloned cDNA to characterize the expression of SCp55 by immunohistochemistry and in situ hybridization. This protein is specific for SC and shows differential expression both during development and between the two SC phenotypes. When they differentiate the protein is first induced in myelinating SC and then in nonmyelinating SC. The nature of this protein together with its differential expression suggests that it is a transcription factor that may have a role in the development of SC.

Folate status during labor: relationship with pregnancy outcome.

OBJECTIVE: The purpose of our study was to determine the folate status of pregnant women at labor, and to detect probable relationships with the gestational age at delivery, the birth weight of the newborns, as well as the mode of the delivery, taking into account any changes in the fetal heart rate (FHR) at labor and, subsequently, operative delivery. METHODS: Maternal serum folate levels were determined using automated fluorometric enzyme-linked assays. Gestational age was determined by ultrasound in the first trimester followed by serial fetal biometry. RESULTS: The results of our study in 101 consecutive pregnant women revealed that the mean (+/-SD) maternal serum concentration of the folate during labor was 12.01 (+/-4.16) ng/ml (range 2.50-23). The mean (+/-SD) gestational age at labor was 38.5 (+/-1.2) weeks (range 35-41 wks) as also the mean (+/-SD) birth weight of the newborns was 3.217 (+/-403) g (range 2,000-4,250 g). CONCLUSIONS: No significant correlation (p>0.05) between folate levels of the maternal serum and gestational age at delivery or birth weight was found. The mode of delivery as a result of probable relationship between operative delivery and maternal serum folate levels was also not found.

Selection of down-regulated sequences along the monocytic differentiation of leukemic HL60 cells.

In order to dissect the molecular mechanisms of monocytic differentiation we have developed a subtractive hybridisation method based on a simplified 'representational difference analysis'. We have selected 16 sequences and confirmed their down-regulation along the TPA-induced monocytic differentiation of HL60 cells. Among these sequences we have identified the alpha-tubulin, the TaxREB protein and two ribosomal protein sequences which had not been previously described as differentially expressed. These results add to our knowledge about the molecules implicated along the monocytic differentiation and growth arrest of leukemic cells and provide a first step in the study of their respective roles.

Complex polysaccharides as PCR inhibitors in feces: Helicobacter pylori model.

A model was developed to study inhibitors present in feces which prevent the use of PCR for the detection of Helicobacter pylori. A DNA fragment amplified with the same primers as H. pylori was used to spike samples before extraction by a modified QIAamp tissue method. Inhibitors, separated on an Ultrogel AcA44 column, were characterized. Inhibitors in feces are complex polysaccharides possibly originating from vegetable material in the diet.

Improved microplate immunoenzymatic assay of PCR products for rapid detection of Mycoplasma pneumoniae.

We developed a microtitre hybridization assay for the detection of polymerase chain reaction (PCR) amplified sequences. For this, cloned Mycoplasma pneumoniae DNA containing a sequence complementary to the PCR products is first covalently bound to microtitre wells. These coated microplates can be stored for several months. Then, an aliquot of the PCR product, labelled with digoxigenin-dUTP during its synthesis is hybridized to the immobilized DNA. The use of a rapid hybridization buffer makes this step very short (5 min). Finally, the hybridization signal is detected by an anti-digoxigenin antibody conjugated with alkaline phosphatase. Compared to Southern or other microplate hybridization techniques, this method is cheaper, involved fewer steps and allows easy handling of a large number of samples. This method was used for detection of M. pneumoniae in a series of clinical specimens.

Polymerase chain reaction using 16S rRNA gene sequences distinguishes the two biovars of Ureaplasma urealyticum.

Several fundamental phenotypic and genotypic differences have separated strains of the genital mycoplasma Ureaplasma urealyticum into two clusters or biovars. However, the lack of an easily performed and unambiguous test to discriminate between them has hampered investigation of the relationship between these biovars and disease. We determined the 16S rRNA nucleotide sequence of U. urealyticum 27, the serovar 3 standard and representative of the parvo biovar (serovars 1, 3, 6, and 14). This sequence was compared with the published sequence of U. urealyticum T960, which is the type strain and the serovar 8 standard and is representative of the T960 biovar which is composed of the 10 intervening serovars. Homology between the two sequences was 98.8%; differences were exploited to provide primers for biovar-specific polymerase chain reactions (PCRs). The results of these reactions placed all 14 serovar standard strains into the correct biovar. The PCRs were also applied to 10 cloned and 8 noncloned isolates that had been serotyped earlier. For 16 of them, we deduced their biovars from the serotyping data and then confirmed them by PCR. One unpredictable isolate and one nonserotypeable isolate were also classified as to biovar. Thus, we have developed a method for biotyping U. urealyticum that is applicable to both laboratory-adapted strains and wild-type isolates and that is appropriate for testing large numbers of clinical isolates. The amplification by the T960 biovar PCR protocol of DNAs from ureaplasmas of animals and certain Mycoplasma species suggested that the parvo biovar has diverged from the mainstream of the evolution of this clade.

Typing of Chlamydia trachomatis by restriction endonuclease analysis of the amplified major outer membrane protein gene.

A procedure was developed for characterization of Chlamydia trachomatis strains by using restriction endonuclease analysis of amplified genes of the major outer membrane protein (MOMP). Reference strains of the 15 serovars (A through K and L1 through L3) and clinical isolates were tested. The nucleotide sequences of the MOMP genes of each of the 15 serovars were arbitrarily constructed by using the sequences of the four variable domains known for each serovar and the constant domains of serovar L1. Computer analysis of these sequences indicated that two restriction digestions performed in parallel, one with AluI and the other with IIpaII, followed by HinfI and EcoRI, would allow the theoretical differentiation of 13 serovars. Serovars Ba and L1 presented the same theoretical restriction profile. Our typing method consisted of polymerase chain reaction amplification of a fragment of about 1,200 bp of the MOMP gene, followed by restriction endonuclease digestion with the aforementioned enzymes. From the 15 serovars, we obtained 14 different patterns; 13 profiles were serovar specific, while serovars B and Ba presented the same pattern. Application of this typing method to C. trachomatis strains isolated from clinical material gave the same results as the immunotyping method for 14 of 17 strains. Furthermore, restriction endonuclease analysis detected differences within a serovar. This method seems to be promising for epidemiological studies.

Specific amplification of a DNA sequence common to all Chlamydia trachomatis serovars using the polymerase chain reaction.

Enzymatic DNA amplification was applied to DNA and elementary bodies of C. trachomatis. Oligonucleotide primers were chosen in a sequence of a conserved domain of the major outer membrane protein to generate the amplification of a 129-base pair fragment. This sequence was amplified in the 15 serovars of C. trachomatis; however, serovar J gave a weaker signal than the others. The specificity was controlled by EcoRI restriction enzyme digestion and Southern analysis using an internal probe of the amplified sequence. No cross-reaction was shown with DNA of 11 other bacteria. Thus, enzymatic DNA amplification by the polymerase chain reaction appears to be a potential tool for the specific detection of C. trachomatis.

Ureaplasma-urealyticum-induced bladder stones in rats and their prevention by flurofamide and doxycycline.

Struvite calculi can be produced in the bladder of Sprague-Dawley male rats after injection of ureaplasmas into the renal medulla. Calculi appear 3 to 6 days after ureaplasma injection. We have studied the inhibitory effect of flurofamide, a potent inhibitor of Ureaplasma urealyticum urease, and doxycycline, on the formation of bladder stones. Flurofamide given orally in five doses (total 125 mg) over 3 days and doxycycline in seven doses (total 20 mg) over 4 days partially prevented stone formation only when given at the time of inoculation. Ureaplasmas disappeared rapidly from the urine. The inhibitory effect of flurofamide was higher than that of doxycycline. However, doxycycline seemed to be efficient when given for a long period (5 weeks).