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D M F Prazeres

Publications and source records attributed to D M F Prazeres.

15 recordsLinked to original sources
PubMed2006

Chemical synthesis and crystallization of the dipeptide AcPheIleNH(2) in TTAB/heptane/octanol reversed micelles.

The chemical synthesis of the dipeptide acetyl phenyl iso-leucin-amide (AcPheIleNH(2)) in tetradecyl trimethyl ammonium bromide (TTAB)/ heptane/octanol/carbonate buffer reversed micelles is described. The co-existence of the surfactant bounded minute water pools within the bulk organic solvent enables the simultaneous solubilization of the polar (IleNH(2)) and apolar (AcPheOEt) substrates, thus enabling the synthesis to take place at the micellar interface. The synthesis was favored by increasing the micellar interface via an increase in water content and surfactant concentration. The best dipeptide yield (87%) was obtained at 32 degrees C, with the largest concentrations of TTAB (200 mM) and water (1100 mM) tested. The low solubility of the dipeptide in the micellar media further led to the formation and growth of needle-like crystals during synthesis, thus enabling the removal of product from solution.

Crystallization↗
PubMed2005

Single base mismatch detection by microsecond voltage pulses.

A single square voltage pulse applied to metal electrodes underneath a silicon dioxide film upon which DNA probes are immobilized allows the discrimination of DNA targets with a single base mismatch during hybridization. Pulse duration, magnitude and slew rate of the voltage pulse are all key factors controlling the rates of electric field assisted hybridization. Although pulses with 1 V, lasting less than 1 ms and with a rise/fall times of 4.5 ns led to maximum hybridization of fully complementary strands, lack of stringency did not allow the discrimination of single base mismatches. However, by choosing pulse conditions that are slightly off the optimum, the selectivity for discriminating single base mismatches could be improved up to a factor approximately 5 when the mismatch was in the middle of the strand and up to approximately 1.5 when the mismatch was on the 5'-end and. These results demonstrate that hybridization with the appropriate electric field pulse provides a new, site-specific, approach to the discrimination of single nucleotide polymorphisms in the sub-millisecond time scale, for addressable DNA microarrays.

Base Pair Mismatch↗
PubMed2005

Optimization of the primary recovery of human interferon alpha2b from Escherichia coli inclusion bodies.

The human interferon alpha2b (hu-IFNalpha2b) gene was cloned in Escherichia coli JM109(DE3) and the recombinant protein was expressed as cytoplasmic inclusion bodies (IB). The present work discusses the recovery of hu-IFNalpha2b IB from the E. coli cells. An optimized protocol is proposed based on the sequential evaluation of recovery steps and parameters: (i) cell disruption, (ii) IB recovery and separation from cell debris, (iii) IB washing, and (iv) IB solubilization. Parameters such as hu-IFNalpha2b purity and recovery yield were measured after each step. The optimized recovery protocol yielded 60% of hu-IFNalpha2b with a purity of up to 80%. The protein was renatured at high concentration after recovery and it was found to display biological activity.

Animals↗
PubMed2005

Chromatography of plasmid DNA.

Liquid chromatography plays a central role in process-scale manufacturing of therapeutic plasmid DNA (pDNA) for gene therapy and DNA vaccination. Apart from its use as a preparative purification step, it is also very useful as an analytical tool to monitor and control pDNA quality during processing and in final formulations. This paper gives an overview of the use of pDNA chromatography. The specificity of pDNA purification and the consequent limitations to the performance of chromatography are described. Strategies currently used to overcome those limitations, as well as other possible solutions are presented. Applications of the different types of chromatography to the purification of therapeutic pDNA are reviewed, and the main advantages and disadvantages behind each technique highlighted.

Chromatography, Liquid↗
PubMed2004

Immobilization and hybridization by single sub-millisecond electric field pulses, for pixel-addressed DNA microarrays.

Single square voltage pulses applied to buried electrodes result in dramatic rate increases for (1) selective covalent bonding (immobilization) of single-stranded DNA (ssDNA) probes to a functionalized thin film SiO(2) surface on a plastic substrate and (2) hybridization of ssDNA to the immobilized probe. DNA immobilization and hybridization times are 100 ns and 10 micros, respectively, about 10(9) times faster than the corresponding passive reactions without electric field. Surface coverage is comparable. Duration, magnitude and slew rate of the voltage pulse are all key factors controlling the rates of ssDNA immobilization and hybridization. With rise times of 4.5 ns, pulses shorter than 1 ms and voltages below 1V are effective. The ssDNA adsorbed on the surface is reoriented by the rapidly changing electric field. This reduces steric barriers and speeds the immobilization and hybridization reactions. These results open the way for pixel-addressed microarrays driven by silicon microelectronics circuits.

Adsorption↗
PubMed2004

An on-chip thin film photodetector for the quantification of DNA probes and targets in microarrays.

A flat microdevice which incorporates a thin-film amorphous silicon (a-Si:H) photodetector with an upper layer of functionalized SiO2 is used to quantify the density of both immobilized and hybridized DNA oligonucleotides labeled with a fluorophore. The device is based on the photoconductivity of hydrogenated amorphous silicon in a coplanar electrode configuration. Excitation, with near UV/blue light, of a single-stranded DNA molecule tagged with the fluorophore 1-(3-(succinimidyloxycarbonyl)benzyl)-4-(5-(4-methoxyphenyl)oxazol-2-yl) pyridinium bromide (PyMPO), results in the emission of visible light. The emitted light is then converted into an electrical signal in the photodetector, thus allowing the optoelectronic detection of the DNA molecules. The detection limit of the present device is of the order of 1 x 10(12) molecules/cm2 and is limited by the efficiency of the filtering of the excitation light. A surface density of 33.5 +/- 4.0 pmol/cm2 was measured for DNA covalently immobilized to the functionalized SiO2 thin film and a surface density of 3.7 +/- 1.5 pmol/cm2 was measured for the complementary DNA hybridized to the bound DNA. The detection concept explored can enable on-chip electronic data acquisition, improving both the speed and the reliability of DNA microarrays.

Base Pairing↗
PubMed2004

Translational features of human alpha 2b interferon production in Escherichia coli.

The yield of human alpha 2b interferon in Escherichia coli was optimized by replacement of low-usage arginine codons located in the mRNA 5' end. The differences observed among the various gene variants suggest that codon usage, Shine-Dalgarno-like sequences, and mRNA secondary structure contribute to the performance of E. coli translation machinery.

Amino Acid Sequence↗
PubMed2003

Hydrophobic interaction chromatography of homo-oligonucleotides on derivatized sepharose CL-6B. Using and relating two different models for describing the effect of salt and temperature on retention.

The effect of salt and temperature on the interaction of homo-oligonucleotides with a derivatized Sepharose CL-6B hydrophobic support has been explained by the application of two different models: the solvophobic theory and the preferential interaction analysis. It has been shown that the two approaches give adequate descriptions of the experimental results. The models were used in order to estimate, respectively the parameter C, which is proportional to the reduction in hydrophobic exposed surface area upon adsorption, and the number of water and salt ions released upon adsorption. It was concluded that the magnitude of these parameters can be strongly influenced by the temperature, the hydrophobicity of the bases of the nucleotides, the molecular mass of the oligonucleotides and the presence of secondary structures. Parameter C was quantitatively related with the number of water molecules and salt ions released upon adsorption. These parameters were found to correlate linearly in cases where structural changes with temperature are not significant.

Models, Chemical↗
PubMed2003

Assessment of purity and quantification of plasmid DNA in process solutions using high-performance hydrophobic interaction chromatography.

A hydrophobic interaction HPLC method was developed for the quantification of plasmid DNA and assessment of its purity in crude Escherichia coli lysates and other process streams. A Phenyl Sepharose Source (Amersham Biosciences) column was used to separate the double-stranded plasmid DNA molecules from the more hydrophobic impurities present in the process streams. The method is rapid (each analysis takes 7 min), reproducible, easy to perform and does not require previous digestion of RNA in samples with RNase or other pre-treatment. Furthermore, it is capable of handling heavily contaminated samples, with less than 5% of plasmid DNA thus constituting a good alternative to other less robust analytical techniques currently in use.

Calibration↗
PubMed2002

Isolation of plasmid DNA from cell lysates by aqueous two-phase systems.

This work presents a study of the partitioning of a plasmid vector containing the cystic fibrosis gene in polyethylene glycol (PEG)/salt (K2HPO4) aqueous two-phase systems (ATPS). The plasmid was extracted from neutralized alkaline lysates using PEG with molecular weights varying from 200 to 8000. The effects of the lysate mass loaded to the ATPS (20, 40, and 60% w/w) and of the plasmid concentration in the lysate were evaluated. The performance of the process was determined by qualitative and quantitative assays, carefully established to overcome the strong interference of impurities (protein, genomic DNA, RNA), salt, and PEG. Plasmid DNA partitioned to the top phase when PEG molecular weight was lower than 400. The bottom phase was preferred when higher PEG molecular weights were used. Aqueous two-phase systems with PEG 300, 600, and 1000 were chosen for further studies on the basis of plasmid and RNA agarose gel analysis and protein quantitation. The recovery yields were found to be proportional to the plasmid concentration in the lysate. The best yields (>67%) were obtained with PEG 1000. These systems (with 40 and 60% w/w of lysate load) were able to separate the plasmid from proteins and genomic DNA, but copartitioning of RNA with the plasmid was observed. Aqueous two-phase systems with PEG 300 concentrated both plasmid and proteins in the top phase. The best system for plasmid purification used PEG 600 with a 40% (w/w) lysate load. In this system, RNA was found mostly in the interphase, proteins were not detected in the plasmid bottom phase and genomic DNA was reduced 7.5-fold.

Blotting, Southern↗
PubMed2002

Hydrophobic interaction chromatography of homo-oligonucleotides on derivatized Sepharose CL-6B. Application of the solvophobic theory.

This work describes the hydrophobic interaction chromatography of homo-deoxyoligonucleotides polyA, polyT and polyU with sizes up to 30 bases on a Sepharose gel derivatized with the 1,4-butanediol diglycidyl ether. The oligonucleotides interacted differently with the column according to the molecular mass, the hydrophobic character of the individual bases, the secondary structure of the molecule and the concentration of ammonium sulphate in the eluent. The retention factor, k', was determined from the chromatographic profiles at different concentrations of ammonium sulphate. A linear relationship between log k' and the concentration of ammonium sulphate in the eluent was found for all oligonucleotides at the higher concentrations (> 1.0 M) of ammonium sulphate. The slope of these plots, termed the hydrophobic interaction parameter, was found to be an increasing function of the number of nucleotides. The same plots reveal that polyA molecules with high molecular mass have lower retention factors when compared with polyT, an observation that was not expected since the hydrophobicity of adenine is higher than that of thymine. This behaviour was due to the existence of secondary structures in polyA, which decrease the exposed hydrophobic area of the molecule.

Chromatography, Gel↗
PubMedDate not supplied

Trends in dengue diagnosis.

The conventional diagnosis of dengue virus infections includes the detection of the virus in serum or tissue samples, both by isolation in culture or through detection of specific viral molecules (genome RNA or dengue antigens) and detection of specific anti-dengue antibodies (serology). Isolation of dengue virus provides the most direct and conclusive approach to diagnosis, despite the demand for high-level equipment, technical skills and manpower. However, it is useless in early diagnosis because several days are required to isolate and classify the virus. Serology, despite being simpler, is not able to afford an accurate early diagnosis in primary infections because 4-5 days are required for the immune system to produce a sufficient amount of antibodies. Moreover, it leads to misleading results in secondary infections owing to cross-reactivity among serotype-specific antibodies and with other flavivirus antibodies. The RT-PCR and other PCR-based techniques are fast, serotype-discriminating, more sensitive and easier to carry out than conventional nucleic-acid hybridisation, but are handicapped by easy sample contamination and high technological demands. Recently, advances in bioelectronics have generated commercial kits and new techniques for detection of dengue antibodies and RNA, based on biosensor technology. Most of them are rapid, easy to operate, reusable, cheap, sensitive and serotype-specific. Nevertheless, their accuracy is still questionable because most still lack validation and standardisation. This review summarises and describes the techniques currently employed and anticipated in the near future for diagnosis of dengue disease.

Antibodies, Viral↗
PubMedDate not supplied

Optimization of isopropanol and ammonium sulfate precipitation steps in the purification of plasmid DNA.

Large-scale processes used to manufacture grams of plasmid DNA (pDNA) should be cGMP compliant, economically feasible, and environmentally friendly. Alcohol and salt precipitation techniques are frequently used in plasmid DNA (pDNA) downstream processing, as concentration and prepurification steps, respectively. This work describes a study of a standard 2-propanol (IsopOH; 0.7 v/v) and ammonium sulfate (AS; 2.5 M) precipitation. When inserted in a full process, this tandem precipitation scheme represents a high economic and environmental impact due to the large amounts of the two precipitant agents and their environmental relevance. Thus, major goals of the study were the minimization of precipitants and the selection of the best operating conditions for high pDNA recovery and purity. The pDNA concentration in the starting Escherichia coli alkaline lysate strongly affected the efficiency of IsopOH precipitation as a concentration step. The results showed that although an IsopOH concentration of at least 0.6 (v/v) was required to maximize recovery when using lysates with less than 80 microg pDNA/mL, concentrations as low as 0.4 v/v could be used with more concentrated lysates (170 microg pDNA/mL). Following resuspension of pDNA pellets generated by 0.6 v/v IsopOH, precipitation at 4 degrees C with 2.4 M AS consistently resulted in recoveries higher than 80% and in removal of more than 90% of the impurities (essentially RNA). An experimental design further indicated that AS concentrations could be reduced down to 2.0 M, resulting in an acceptable purity (21-23%) without compromising recovery (84-86%). Plasmid recovery and purity after the sequential IsopOH/AS precipitation could be further improved by increasing the concentration factor (CF) upon IsopOH precipitation from 2 up to 25. Under these conditions, IsopOH and AS concentrations of 0.60 v/v and 1.6 M resulted in high recovery (approximately 100%) and purity (32%). In conclusion, it is possible to reduce substantially the mass of precipitation agents used without affecting recovery, if a small concession is made regarding purity. This directly translates into an improvement of the process economics and in a reduction of the environmental impact of the process.

2-Propanol↗