Biomedical subjects

Denise Thomas

Publications and source records attributed to Denise Thomas.

3 recordsLinked to original sources

PubMed2005

Preparative-scale fractionation by isoelectric trapping under nondenaturing conditions: separation of egg white protein isoforms on a modified Gradiflow unit.

pH-biased isoelectric trapping was used to separate proteins from egg white at the preparative level (80 mg), into discrete protein fractions based on isoelectric point. The problems of isoelectric precipitation that are common for the separation of complex protein mixtures under isoelectric conditions were mitigated by using single-component isoelectric buffers within the sample separation compartments. This combined with the mild process conditions of the Gradiflow unit that was modified for binary isoelectric trapping separations, ensured that biological activity was maintained. This was verified by measurement of the trypsin protease inhibitory activity of the extract and separated fractions. Furthermore, the high resolving power of this system under preparative conditions was demonstrated by separation of three protein isoforms using isoelectric membranes with differences of 0.025 pH units from each other.

Animals↗

PubMed2005

Monitoring of a pharmaceutical nanomilling process using grating light reflection spectroscopy.

An optical diagnostic method, grating light reflection spectroscopy (GLRS), has been demonstrated for the in situ monitoring of properties of heterogeneous matrices in industrial processes. The technique is based on measurements near the critical points of intensity and phase in waves reflected from a transmission diffraction grating in contact with a diagnostic sample. The features contained in the reflection spectrum near these thresholds allow for the simultaneous determination of the real and imaginary parts of the dielectric function of the sample. Using these data, the milling progress of highly concentrated fluid suspensions is observed as the material is milled from approximately 40 mm to 160 nm in diameter. A theoretical model that closely resembles experimentally determined spectra was constructed and applied in combination with principal components analysis (PCA) to demonstrate that GLRS can be used to closely monitor changes in the mean particle size of the nanomilled drug product.

Algorithms↗

PubMed2002

A Janus splicing regulatory element modulates HIV-1 tat and rev mRNA production by coordination of hnRNP A1 cooperative binding.

Retroviral protein production depends upon alternative splicing of the viral transcript. The HIV-1 acceptor site A7 is required for tat and rev mRNA production. Production of the Tat transcriptional activator is highly controlled because of its apoptotic properties. Two silencer elements (ESS3 and ISS) and two enhancer elements (ESE2 and ESE3/(GAA)3) were previously identified at site A7. hnRNP A1 binds ISS and ESS3 and is involved in the inhibitory process, ASF/SF2 activates site A7 utilisation. Here, by using chemical and enzymatic probes we established the 2D structure of the HIV-1(BRU) RNA region containing site A7 and identified the RNA segments protected in nuclear extract and by purified hnRNP A1. ISS, ESE3/(GAA)3 and ESS3 are located in three distinct stem-loop structures (SLS1, 2 and 3). As expected, hnRNP A1 binds sites 1, 2 and 3 of ISS and ESS3b, and oligomerises on the polypurine sequence upstream of ESS3b. In addition, we discovered an unidentified hnRNP A1 binding site (AUAGAA), that overlaps ESE3/(GAA)3. On the basis of competition experiments, hnRNP A1 has a stronger affinity for this site than for ESS3b. By insertion of (GAA)3 alone or preceded by the AUA trinucleotide in a foreign context, the AUAGAA sequence was found to modulate strongly the (GAA)3 splicing enhancer activity. Cross-linking experiments on these heterologous RNAs and the SLS2-SLS3 HIV-1 RNA region, in nuclear extract and with recombinant proteins, showed that binding of hnRNP A1 to AUA(GAA)3 strongly competes the association of ASF/SF2 with (GAA)3. In addition, disruption of AUA(GAA)3 demonstrated a key role of this sequence in hnRNP A1 cooperative binding to the ISS and ESS3b inhibitors and hnRNP A1 oligomerisation on the polypurine sequence. Thus, depending on the cellular context ([ASF/SF2]/[hnRNP A1] ratio), AUA(GAA)3 will activate or repress site A7 utilisation and can thus be considered as a Janus splicing regulator.

Alternative Splicing↗