AABRE: NETWORKING IN PUERTO RICO

PR-AABRE Researcher: Iván Ferrer-Rodríguez, Ph.D, Department of Natural Sciences and Mathematics, Inter American University of Puerto Rico, Bayamón Campus

Cluster: Molecular Medicine

Collaborator: Fidel Zavala, M.D. (Johns Hopkins University)

Mentor: Adelfa Serrano, Ph.D. (UPR, Medical Sciences Campus)

Project Title: Identification and Expression Analysis of ABC Genes in Plasmodium yoelii

IN THE PAST CENTURY, medicine has triumphed over an impressive array of infectious diseases, increasing the life expectancy of the average American by about 30 years. Antibiotics and vaccines against parasites, bacteria, and viral illnesses, as well as pesticides that kill carriers of many fatal diseases, are largely responsible for our longer, healthier lives. According to the World Health Organization (WHO), Puerto Rico was the first country to be officially certified as malaria free. Early in the 1960s, the optimistic John F. Kennedy administration vowed to eradicate malaria completely. Today, however, the incidence of malaria has increased, despite the many drugs that were developed to fight it. About 300 million people are infected and about 1.5 million die annually from malaria, according to the WHO. Children and Africans are affected the most; every thirty seconds one child dies from malaria. The miracle antimalarial drugs just aren’t working anymore.

“Parasites become resistant to drugs. The malaria parasites have become resistant to antimalarial drugs, and malaria-carrying mosquitoes have become resistant to insecticides,” says biologist Iván Ferrer. Ferrer and his research team are trying to find out why and how the malaria parasites become resistant to antimalarial drugs, specifically to the quinoline-derived drugs. This knowledge would help the researchers in efforts to revert resistance to older antimalarial drugs or new drugs that kill resistant parasites.

Ferrer says he hopes his team can discover a way to make the old drugs effective again because they worked better than newer drugs, caused fewer side effects, and are cheaper. Very few new antimalarial drugs are available because malaria occurs predominantly in countries that cannot afford to buy the drugs, making antimalarial drug development unprofitable, he says. “In addition, parasites develop multidrug resistances—once they become resistant to one drug, they become resistant to others.”

The researcher uses malaria–infected rodents as a model because the parasites that infect the rodents are similar to the parasites that infect humans, and they are susceptible to the same drugs. “From our previous experiments with the parasites, we know that certain genes could be involved in drug resistance, for example, the genes that code for the ABC superfamily of transporter proteins. We are focusing on two genes, and we think that the genes are working together to confer resistance. We’re trying to characterize these genes.” Student researchers are sequencing these two genes in drug sensitive and resistant lines of the parasite to see if mutations are responsible for resistance.

By comparing the genes of drug sensitive parasites with those that are drug resistant, the researchers hope to pinpoint—with the help of computer assisted analysis of gene sequencing—the spot on the gene responsible for resistance. Other mechanisms of drug resistance, such as gene amplification and increased expression, will also be evaluated. The researchers are using Quantitative PCR, Real-time PCR, Southern blot, Northern blot, and Western blot to characterize the genes. Ferrer said that he focused his study on quinoline-derived drugs because they worked the best before resistances developed. “Chloroquine is like the penicillin of malaria.”

iferrer@bc.inter.edu

POLYMERASE CHAIN REACTION (PCR) PCR is a quick way to generate unlimited copies of any fragment of DNA. The genetic material of every living organism possesses unique sequences of nucleotide building blocks specific to its own species, making it possible to trace genetic material back to its origin to determine species and often which particular member of that species it came from. PCR begins with a template molecule, the DNA or RNA to be copied, and two primer molecules. The primers are short chains of the four chemical components that make up any strand of genetic material. Polymerases, enzymes present in all living things, will copy, proofread, and correct genetic material. The three steps in a PCR are repeated for 30 or 40 cycles with an automated cycler that quickly heats and cools tubes containing the reaction mixture. The first step is Denaturation. When heated to 94°C, the double strand of the DNA helix separates into two single strands, and all enzymatic reactions stop. Then the tube is cooled to 54°C for the Hybridization or Annealing step in which ionic bonds are formed and broken between the primer and the template. The more stable bonds—with primers that fit exactly—last a little longer; and on that little piece of double stranded DNA (template and primer), the polymerase attaches and starts to copy the template. In the Extension step, the tube is heated to 72°C. The polymerase can read a template strand and very quickly match it with complementary nucleotides. The result is duplication: two new helixes replace the first, and each is composed of one original strand plus its newly formed complementary strand.

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