Clinical cases and Situational tasks

106. Antibiotic resistance develops because these drugs are overused in medical practice and in livestock feeds. Suggest the mechanism by which this extensive use promotes antibiotic resistance.

Answer: When antibiotics are used in large quantities, the bacterial cells that possess resistance genes (acquired through mutations or through intermicrobial DNA transfer mechanisms) survive and even flourish. Because of antibiotic use, which acts as a selection pressure, resistant organisms become the dominant cells in their ecological niche.

107. Briefly outline the basic principles of PCR. Calculate the degree of amplification attained by 15 PCR cycles.

Answer: PCR begins by adding polymerase, primers to a heated sample of the target DNA. As the mixture cools, the primers attach to their complementary sequence on either side of the target sequence. Each strand then serves as a template for DNA replication. On the end of this process, referred to as a cycle, the copies of the target sequence have been doubled. The process can be repeated indefinitely, synthesizing an extraordinary number of copies. After 15 replications 2¹⁵ copies have been produced.

108. List and describe properties of the genetic code.

Answer: The genetic code is degenerate (several codons have the same meaning), specific (each codon specifies only one amino acid), universal (with a few exceptions each codon always specifies the same amino acid). In addition genetic code is nonoverlapping and without punctuations (i.e., mRNA is read as continuous coding sequence).

109. A 48-year-old man has had a lengthy history of skin cancer. In the past 6 years he has had over 30 neoplasms removed from sun-exposed areas and has been diagnosed with xeroderma pigmentosum. Which disorder best describes the enzymatic defect in patients with xeroderma pigmentosum? Explain your answer.

Answer: Xeroderma pigmentosum is a genetic disease in which the ability to remove pyrimidine dimers caused by exposure to UV light is impaired. The mechanism used to remove these pyrimidine dimmers (also used to repair DNA that has formed adducts with carcinogenic compounds) is excision repair. The enzymes used in this repair mechanism cleave the affected strand on either side of damaged nucleotides. The oligonucleotide containing the damaged nucleotides is removed and the gap is filled in by DNA polymerase and DNA ligase.

110. A 32-year-old female is being treated with methotrexate for a recently diagnosed choriocarcinoma of the ovary, and presents with complaints of oral mucosal ulcers. The patient recalls being advised not to take folate-containing vitamins during therapy. An uncomplicated surgical exploration was performed 5 weeks ago with removal of the affected ovary. The patient has been taking methotrexate for 2 weeks and has never had any of the above symptoms before. On examination, patient was afebrile and appeared ill. Several mucosal ulcers were seen in her mouth. The patient also had some upper abdominal tenderness. Her platelet count is decreased at 60,000/mm³ (normal 150,000 to 450,000/mm³). What is the most likely etiology of her symptoms? What is the biochemical explanation of her symptoms? Explain the mechanism of methotrexate action and its cytotoxicity.

Answer: Likely cause of her symptoms: Side effects of methotrexate (antimetabolite chemotherapy) affecting rapidly dividing cells such as oral mucosa. Biochemical explanation of her symptoms: Related to effects of methotrexate on cell cycle of all cells (particularly rapidly dividing cells). Folate antagonists inhibit dihydrofolate reductase (tetrahydrofolate needed for purine synthesis). Mechanism of action: Chemotherapeutic agents are used to treat various types of cancers. Although some are specific for cancer cells, most chemotherapeutic agents are toxic for both normal and cancer cells. Methotrexate acts as a folate antagonist, affecting DNA synthesis. Because cancer cells divide faster than normal cells, a higher proportion of these neoplastic cells will die. Nevertheless, normal cells that also are rapidly dividing, such as the gastrointestinal mucosa, the oral mucosa, and the bone marrow cells, may be affected. The patient was advised to avoid folate during therapy, since folate would be an “antidote,” and would allow the cancer cells to escape cell kill.

111. A 46-year-old male presents to the emergency department with severe right toe pain. The patient was in usual state of health until early in the morning when he woke up with severe pain in his right big toe. The patient denies any trauma to the toe and no previous history of such pain in other joints. On examination, he was found to have a temperature of 38.2°C (100.8°F) and in moderate distress secondary to the pain in his right toe. The right big toe was swollen, warm, red, and exquisitely tender. The remainder of the examination was normal. Synovial fluid was obtained and revealed rod- or needle-shaped crystals that were negatively birefringent under polarizing microscopy, consistent with gout. What is the likely diagnosis? How would you make a definite diagnosis? What is the pathophysiology of this disorder?

Answer: Diagnosis: Gouty arthritis. Confirming diagnosis: Demonstration of the presence of the monosodium urate crystals within the synovial leukocytes or in material derived from tophi under polarizing microscopy. Pathophysiology: Increased conversion of purine bases to uric acid or a decreased excretion of uric acid by the kidney. Elevated levels of the insoluble uric acid result in precipitation of urate crystals in the joints.

112. Inherited defects in components of purine catabolism and salvage are associated with various conditions and syndromes. The enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT) is a key enzyme in the purine salvage pathway. It is responsible for reforming IMP and GMP from hypoxanthine and guanine, respectively. In this manner purine bases are salvaged back into the purine nucleotide pool. What genetic defects that lead to the loss of HGPRT activity are the primary cause for the following conditions?

Answer: Lesch-Nyhan syndrome results from an inherited deficiency in HGPRT. This syndrome is associated with mental retardation and self-destructive behavior, which may be associated with inadequate production of purine nucleotides through the salvage pathway in certain neuronal cells. In addition, Lesch-Nyhan patients have gout resulting from the inability to salvage purine bases, which leads to increased levels of uric acid. However, most patients with gout do not have a defect in HGPRT but have hyperuricemia resulting from a number of factors, including diet.