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Evidence suggests that most chemotherapeutic agents are less effective as treatment in patients with estrogen receptor-negative (ER-) breast carcinomas compared to those with estrogen receptor-positive (ER+) breast carcinomas. IntroductionCancer of the breast is the most commonly diagnosed non-skin cancer and second leading cause of cancer-related deaths in women. Moreover, African American Women (AAW) is disproportionately diagnosed with ER- breast cancer compared to their white counterparts. Agarose gel electrophoresis of genomic DNA samples of VA and spinach were digested with restriction enzyme Bam HI. Agarose gel electrophoresis of genomic DNA samples of VA and spinach were digested with restriction enzyme Hind III. Novel therapies effective against ER- breast carcinomas are urgently needed to ameliorate the health disparity. An estimated 178,000 women will be diagnosed with invasive breast cancer and 40,460 women will die from the disease this year in the U.S.
Agarose gel electrophoresis of genomic DNA samples of VA and spinach were digested with restriction enzyme Eco RI. However, the anti-proliferative activities of VA in either ductal or ER- carcinoma cells have not been characterized. Tamoxifen (TAM), an anti-estrogen drug, is one of the most effective chemotherapies for ER+ breast carcinomas [3]. A full understanding of a gene, or the entire set of genes in a genome, requires that they be isolated and then studied intensively.
Paclitaxel or Taxol (TAX), an anti-microtubule agent; is effective against estrogen receptor-negative (ER-) breast carcinomas [3–4].
Once a gene is Oin handO, in principal one can determine both its biochemical structures and its function(s) in an organism.
Estrogen Receptor A Mediates Breast Cancer Cell Resistance to Paclitaxel through Inhibition of Apoptotic Cell Death. One of the goals of biochemistry and molecular genetics is to assign particular functions to individual or composite structures. This chapter covers some of the techniques commonly used to isolate genes and illustrates some of the analyses that can be done on isolated genes.
One of the reported reasons for the breast cancer disparity is that AAW are more likely to be diagnosed with ER- breast cancer (a more aggressive breast cancer, with less treatment options) than other ethnic groups [5–7].
However, methods to isolate genes were not developed until the 1960Os, and the were applicable to only a few genes.
Isolation of DNA from dried VA leaves yielded approximately 12.2 and 1 kbp genomic DNA that were Eco RI-insensitive but Hind III and Bam HI-sensitive. Therefore, there is an urgent need for the discovery and development of agent(s) efficacious against ER- breast cancer to close or eliminate the breast cancer disparity gap.
Racial disparities in female breast cancer in South Carolina: clinical evidence for a biological basis. These pieces of information may be used to enhance the safety of medicinal botanical VA through authentication, and adulteration detection.
Vernonia amygdalina (VA) is increasingly emerging as a very strong candidate for breast cancer treatment. This technique enabled researchers to isolate any gene from any organism from which one could isolate intact DNA (or RNA). The full potential to provide access to all genes of organisms is now being realized as full genomes are sequenced. One of the by-products of the intense investigation of individual DNA molecules after the advent of recombinant DNA was a procedure to isolate any DNA for which one knows the sequence. It grows in several parts of Africa, including the tropics and particularly South Africa, Zimbabwe and Nigeria [8–10].
This technique, called the polymerase chain reaction (PCR), is far easier than traditional molecular cloning methods, and it has become a staple of many laboratories in the life sciences. African-American race is associated with a poorer overall survival rate for breast cancer patients treated with mastectomy and doxorubicin-based chemotherapy. By isolating these hybrid bacteriophage, the DNA for the bacterial gene could be recovered in a highly enriched form. These compounds isolated from VA extracts, using various solvents of different polarity indexes, have been attributed to specific biological activities. For example: the antiplasmodial (anti-malarial activity) of VA extracts may be related to the presence of flavonoids, saponins, alkaloids [23].

Some studies have associated coumarines and flavonoids in most plants with antitumor activities in humans [26]. Other cancer-fighting agents in VA extracts may include sesquiterpene lactones (SLs) [17–18] and edotides [19].Taken together, there is compelling evidence to show that VA supplementation or therapy may benefit cancer patients.
However, the challenge is that antagonistic relationships exist between conventional medicine (CM) and traditional medicine (TM) practitioners. Induction of the lysogen will result in excision of the prophage and multiplication to produce many progeny, i.e. African indigenous plants with chemotherapeutic potentials and biotechnological approach to the production of bioactive prophylactic agents. First, the contention is that conventional drugs are standardized and chemically-defined; the quantities and structures of the active ingredients are known. The bacteria carrying the prophage show no obvious signs of the phage (except immunity to superinfection with the same phage, covered later in Part Four), but when induced (e.g.
In contrast, traditional medicines are often native or non-purified botanical extracts with limited knowledge of their chemical compositions.
Second, the issues of authentication and quality control must also be improved if TM is to gain more recognition.
In this regard, we have used sample scale-up, extraction, solvent partitioning, column fractionation, profiling with an ultra violet (UV) detector and individual component spectroscopy using nuclear magnetic resonance (NMR), and the combination of HPLC and thin layer chromatography (TLC) techniques to purify VA extracts [under revision].
The objectives of the present studies are to: develop some genetic markers for the VA leaf using restriction enzyme digestion assay for authentication and adulteration detection, and to assess the anticancer activity of VA on ductal breast carcinoma cells. The bacterial gene in the transducing phage has been separated from the other 4000 bacterial genes (in E. Ethnopharmacology of some of the asteraceae family used in the Nigerian traditional medicine. By isolating large numbers of the transducing phage, the phage DNA, including the bacterial genes, can be obtained in large quantities for biochemical investigation.
Imprecise excision from any of those locations generates a particular transducing phage, carrying a short sections of the bacterial genome adjacent to the integration site.
The leaves were rinsed with distilled water and spread out evenly on galvanized-wire screens with the edges bent upward 2 inches on all sides. The physiological function of restriction endonucleases is to serve as part of system to protect bacteria from invasion by viruses or other organisms. The galvanized-wire screens were placed in a specially-constructed dryer and heated to 130–140 ° F for complete dryness within 4 h. The mixture was then filtered through clean white gauze to remove the particulate matter before filtration through a 0.45- ?m filtration unit for sterilization. Hepatoprotective and antioxidant activities of Vernonia amygdalina on acetaminophen-induced hepatic damage on mice. They can be ligated onto any blunt‑ended molecule, thereby generating a new restriction cleavage site on the ends of the molecule.
Thus by incubating each DNA fragment with the appropriate dNTP and terminal deoxynucleotidyl transferase, one can add complementary homopolymers to the ends of the DNAs that one wants to combine. Vernodalin and Vernomygdin, two new cytotoxic sesquiterpene lactones from Vernonia amygdalina Del. Bitter steroid glucosides, Vernoniosides A1, A2,A3 and related B1 from possible medicinal plant-Vernonia amygdalina used by wild chimpanzees. Discovery of water soluble anticancer agents (Edotides) from vegetables found in Benin City, Nigeria.
BT-549 cells were allowed to grow to 60% confluence before stimulating the cells with either VA or paclitaxel for 18 h. Time and dose-dependent modulation of phase 1 and phase 2 gene expression in response to treatment of MCF-7 cells with a natural anti-cancer agent. Replication is usually dependent on host functions, such as DNA polymerases, but regulation of plasmid replication is distinct from that of the host chromosome. All incubations were terminated by aspirating the RPMI medium and doing triplicate washes with 2 mL of cold PBS to remove excess [3H] thymidine.
Upon solubilization, 1 mL of cell solution and 5 mL of scintillation cocktail were mixed thoroughly in each vial.
A novel natural inhibitor of extracellular signal-regulated kinases human breast cancer cell growth.

Plasmid pBR322 carries two antibiotic resistance genes, each derived from different transposons. The pUC plasmids (named for plasmid universal cloning) and plasmids derived from them use a rapid screen for inactivation of the b-galactosidase gene to identify recombinants (Fig. Digestion was done for each reaction that consisted of 10X buffer, restriction enzyme, DNA sample, and water.
In Vitro Antiplasmodial activity of extracts and fractions of seven medicinal plants used in the Democratic Republic of Congo. The pUC vector has the b‑galactosidase gene {actually only part of it, but enough to form a functional enzyme with the rest of the gene that is encoded either on the E.
DNA that has the cohesive ends of l can be packaged in vitro into infective phage particles.
Being in a viral particle brings the efficiency of infection reliably over 108 plaque forming units per mg of recombinant DNA. Some other bacteriphage vectors for cloning are derived from the virus M13.
It has been modified to carry a gene for b‑galactosidase as a way to screen for recombinants. Treatment of the genomic DNA with BamHI produced a smaller band of approximately 1.1 kbp (Fig.
Phagemids are plasmids (with the modified, high-copy number ColE1 origin) that also have an M13 origin of replication. EcoR1 digestion of VA DNA resulted in fainter 12.2 and 1 kbp bands which may suggest an incomplete digestion by EcoR1. Fragments in a similar size range are also cloned into bacterial artificial chromosomes (BACs), which are derived from the F-factor (Fig. BACs have become one of the most frequently used vectors for large inserts in genome projects.
By amplifying a designated segment of DNA, it provides a means to isolate that particular DNA segment or gene. This method requires knowledge of the nucleotide sequence at the ends of the region that you wish to amplify. Once that is known, one can make large quantities of that region starting with miniscule amounts of material, such as the DNA within a single human hair. With the availability of almost complete or complete sequences of genomes from many species, the range of genes to which it can be applied is enormous. The applications of PCR are numerous, from diagnostics to forensics to isolation of genes to studies of their expression. The power of PCR lies in the exponential increase in amount of DNA that results from repeated cycles of DNA synthesis from primers that flank a given region, one primer designed to direct synthesis complementary to the top strand, the other designed to direct synthesis complementary to the bottom strand (Fig. Each cycle consists of a denaturation step at a temperature higher than the melting temperature of the duplex DNA (e.g. 95oC ), then an annealing step at a temperature below the melting temperature for the primer-template (e.g.
55oC), followed by extension of the primer by DNA polymerase using dNTPs provided in the reaction. Thermocylers are commercially available for carrying out many cycles quickly and reliably. The template supplied for the reaction is the only one availablein the first cycle, and it is still a major template in the second cycle. Thus in 21 cycles, one can achieve a million-fold increase in the amount of that DNA (assuming all cycles are completely efficient). These have been isolated from bacteria that grow in hot springs, such as those found in Yellowstone National Park, such as Thermus aquaticus. The Taq polymerase from this bacterium will retain activity even at the high temperatures needed for melting the templates, and it is active at a temperature between the melting and annealing temperature. Hundreds of thousands of ESTs are available, and contain at part of the DNA sequence from many, if not most, human genes. Use of this method to isolate the receptor for the glycoprotein hormone erythropoietin is illustrated in Fig.

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