Abstract: The present invention is the development of a simple and specific quantitative method for the determination of P. falciparum DNA in malaria that involves the direct detection of the highly 42-kDa conserved C-terminal region of P. falciparum merozoite surface protein (MSP1) gene. This procedure entails the amplification of the 42-kDa C-terminal region of the MSP1 gene by using the PCR technique in the presence of digoxigenin-11-dUTP and the synthesis of the specific biotin labeled nucleotide probes directed to the 42-kDa C-terminal region of the MSP1 gene. These specific probes are then used in the Enzyme Linked Immunosorbent Assay (ELISA) for the quantitative determination of the 42-kDa C-terminal region of the MSP1 gene which leads to the quantitative determination of P. falciparum DNA in malaria for quantitative diagnostic purpose as well as for monitoring the efficacy of antimalarial treatment.

Abstract: To maximize the yield of protein from a baculovirus system, optimal infection of the host cell culture must be achieved; in order to obtain such optimal infection, the titer of the virus inoculation must be known. The present invention is the development of a simple, rapid, and universally applicable titration method that involves the direct detection of the viral DBP gene derived from AcNPV (AcNPV DBP) as a target for quantitative titer determination of baculovirus and the use of biotin specific probes directed to viral DBP gene. The procedure entails the amplification of the AcNPV DBP gene by using the PCR technique in the presence of digoxigenin-11-dUTP from the negative control (non-infected) and infected samples, and the synthesis of the specific biotin label nucleotide probes directed to the AcNPV DBP gene.

Abstract: The present invention is the development of a simple and specific quantitative method for the determination of P. falciparum DNA in malaria that involves the direct detection of the highly 42-kDa conserved C-terminal region of P. falciparum merozoite surface protein (MSP1) gene. This procedure entails the amplification of the 42-kDa C-terminal region of the MSP1 gene by using the PCR technique in the presence of digoxigenin-11-dUTP and the synthesis of the specific biotin label nucleotide probes directed to the 42-kDa C-terminal region of the MSP1 gene. These specific probes are then used in the Enzyme Linked Immunosorbent Assay (ELISA) for the quantitative determination of the 42-kDa C-terminal region of the MSP1 gene which leads to the quantitative determination of P. falciparum DNA in malaria for quantitative diagnostic purpose as well as for monitoring the efficacy of antimalarial treatment.

Abstract: Autographa californica nucleopolyhedrovirus (AcNPV) is a baculovirus widely employed as a vector for the expression of foreign genes and pest control. Although baculoviruses efficiently replicate in the nuclei of arthropod cells, the dynamics and mechanism of DNA replication within the infected cell are still poorly understood. Thus, to study such viral DNA replication, the availability of peptide ligands specific for DNA-binding protein (DBP) is needed. This work resulted in the selection of peptide ligands specifically binding to DBP for AcNPV from the FliTrx™ random peptide display library, which entails the amplification, cloning of the DBP gene from AcNPV, the construction of the expression plasinid for DBP, and the expression and purification of the recombinant His.Tag AcNPV DBP which was used as a target molecule for the selection of the peptide ligands. The affinity of peptide ligands was measured by ELISA procedures.

Abstract: Beta-amyloid peptide (?A) is a major fibrillar component of neuritic plaques in Alzheimer's disease brains and is related to the pathogenesis of the disease. ?A generation depends on proteolytic cleavage of the amyloid precursor protein (APP). The present invention is a new procedure for the cloning of human ?A precursor protein gene (human APP gene) based on the reverse transcription (RT) and the polymerase chain reaction (PCR). This procedure for cloning human APP gene by means of RT-PCR reactions is cost-effective, not time-consuming, and is suited for any laboratory. The present invention also includes a new procedure for the construction of expression plasmids, a/ using the pFastBac™ HTb and the pBlueBacHis2 A transfer vectors for the purpose of obtaining human APP in insect cells; and b/ using the pET-28a (+) transfer vector for the purpose of obtaining human APP in bacteria.

Abstract: Spinal muscular atrophy (SMA) is a lethal autosomal recessive disease. The gene most highly associated with SMA is the survival motor neuron (SMN) gene. The present invention is a new procedure for the cloning of human SMN, the SMA determining gene, based on the reverse transcription (RT) and the polymerase chain reaction (PCR). This procedure for cloning human SMN gene by means of RT-PCR reactions is cost-effective, not time-consuming, and is suited for any laboratory. The present invention also includes a new procedure for the construction of expression plasmids, a/ using the pFastBac™ HTb and the pBlueBacHis2 A transfer vectors for the purpose of obtaining human SMN protein in insect cells; and b/ using the pET-28a (+) transfer vector for the purpose of obtaining human SMN protein in bacteria. The present invention makes it easier to obtain full-length SMN protein which is valuable for biochemical and biological analyses that may elucidate the molecular mechanism of SMA.

Abstract: The present invention concerns the development of a quantitative method for the molecular diagnosis of autosomal recessive spinal muscular atrophy (SMA) by measuring the amount of cytosolic mRNA from human muscle cells. Both the procedure using radioactive material and the Enzyme-Linked Immunosorbent Assay (ELISA) nonradioactive method were developed using 32P-dCTP labeled and biotinylated nucleotide probes. The results obtained demonstrate that the measurement of mRNA could be used as a quantitative method for the molecular diagnosis of SMA. There was a perfect concordance of the results obtained between the procedure using radioactive material, the ELISA method and the single strand conformation polymorphism (SSCP) analysis regarding the negative and positive SMA samples.

Abstract: Beta-amyloid peptide (&bgr;A) is a major fibrillar component of neuritic plaques in Alzheimer's disease (AD) brains and is related to the pathogenesis of the disease. The present invention provides a method using Poly-L-Lysine to dissolve preformed &bgr;A fibrils in vitro. Its efficiency is instantaneous. Poly-L-Lysine offers the simplest and most effective way to dissolve preformed &bgr;A fibrils. The method of this present invention can be used as a universal dissolver of all types of oligomeric &bgr;-sheet conformation, precursor of the fibrils. Poly-L-Lysine may also be useful as a future universal therapeutic agent to prevent and/or retard amyloidogenesis in vivo AD and other types of amyloid related disorders.

Abstract: The present invention concerns the development of a quantitative method for the molecular diagnosis of autosomal recessive spinal muscular atrophy (SMA) by measuring the amount of cytosolic mRNA from human muscle cells. Both the procedure using radioactive material and the Enzyme-Linked Immunosorbent Assay (ELISA) nonradioactive method were developed using 32P-dCTP labeled and biotinylated nucleotide probes. The results obtained demonstrate that the measurement of mRNA could be used as a quantitative method for the molecular diagnosis of SMA. There was a perfect concordance of the results obtained between the procedure using radioactive material, the ELISA method and the single strand conformation polymorphism (SSCP) analysis regarding the negative and positive SMA samples.

Abstract: Beta-amyloid peptide (&bgr;A) is a major fibrillar component of neuritic plaques in Alzheimer's disease (AD) brains and is related to the pathogenesis of the disease. The present invention provides a method using Poly-L-Lysine to dissolve preformed &bgr;A fibrils in vitro. Its efficiency is instantaneous. Poly-L-Lysine offers the simplest and most effective way to dissolve preformed &bgr;A fibrils. The method of this present invention can be used as a universal dissolver of all types of oligomeric &bgr;-sheet conformation, precursor of the fibrils. Poly-L-Lysine may also be useful as a future universal therapeutic agent to prevent and/or retard amyloidogenesis in vivo AD and other types of amyloid related disorders.