Exam 3: Biology

Passage Quantitative fluorescence recovery after photobleaching (FRAP) is used to study the movement of molecules in live cells.  During FRAP, the fluorescence of a green fluorescent protein (GFP)-tagged molecule is first measured and then photodestroyed in targeted cell regions.  Researchers then assess the time course for fluorescence recovery, which is an indicator of molecular mobility of the GFP-tagged protein into the photodestroyed region.  The average time required to recover 80% of the pre-bleach fluorescence of protein histone 1 (H1) fused to GFP (H1-GFP) in the photodestroyed region is given as t80.FRAP was used to analyze the mobility of the architectural H1 alone and in the presence of high-mobility group (HMG) proteins.  HMG proteins are dynamic modifiers that have been shown to have opposite effects but similar binding sites on chromatin structure. Figure 1  Unique chromatin binding motifs of HMG proteinsDuring analysis, HMGA1 and HMGB1 were microinjected into the cytoplasm of mouse embryonic fibroblast cells expressing H1-GFP.  FRAP was performed on euchromatin and heterochromatin.  Euchromatin and heterochromatin domains were identified as regions weakly and strongly stained by H1-GFP, respectively.  The relative intensity of H1-GFP fluorescence in euchromatin and heterochromatin of uninjected and injected cells was assessed, and t80 results are shown in Table 1.Table 1  Effect of HMGB1 and HMGA1 on H1-GFP Mobility Adapted from Catez F, Yang H, Tracey KJ, Reeves R, Misteli T, Bustin M. Network of dynamic interactions between histone H1 and high-mobility-group proteins in chromatin. Mol Cell Biol. 2004;24(10):4321-8. -The experimental design described in the passage allowed researchers to do which of the following?Assess whether structural chromatin organization influences H1 mobilityAssess H1 and GFP competition at different levels of DNA compactionEvaluate if HMG proteins compete with H1 for chromatin-binding sites

One function of catecholamines released by the adrenal glands during times of stress is to:

Passage The adrenal glands are divided into two anatomically distinct regions, the adrenal medulla and the adrenal cortex.  The adrenal medulla is a collection of irregularly shaped cells located in the interior portion of each adrenal gland.  In response to changing serum levels of corticosteroid hormones, medullary cells synthesize and secrete the catecholamines norepinephrine and epinephrine.  Norepinephrine and epinephrine are water-soluble compounds, and their actual secretion into the bloodstream is stimulated by acetylcholine (ACh)-mediated signaling in the sympathetic component of the autonomic nervous system.  Figure 1 shows the enzyme-mediated synthesis of norepinephrine and epinephrine. Figure 1The adrenal cortex is the outer portion of the adrenal gland and is composed of cells that synthesize and secrete lipophilic hormones derived from cholesterol.  Hormones produced by the adrenal cortex include the principal glucocorticoid hormone (cortisol) and the mineralocorticoid hormone (aldosterone).The hypothalamus and pituitary gland regulate and control the endocrine functions of the adrenal cortex (Figure 2).  Secretion of corticotrophin-releasing hormone (CRH) from the hypothalamus promotes the synthesis and release of adrenocorticotrophic hormone (ACTH) by the pituitary gland.  ACTH acts on adrenal cortex cells to promote the growth of cortical tissue and to increase the synthesis and secretion of glucocorticoids.  In addition to its body-wide functions as the principal stress hormone, cortisol also regulates adrenal activity by inhibiting CRH release from the hypothalamus and ACTH release from the pituitary. Figure 2 -Hormones secreted by the adrenal medulla most likely influence energy metabolism by:

Passage Eukaryotic organelles such as mitochondria and chloroplasts are believed to be relics of formerly free-living prokaryotes.  The transition from a hypoxic (low O₂) to an oxic atmosphere (21% O₂) is said to have enabled primitive eukaryotic anaerobes to engulf ancient aerobic prokaryotes and consequently acquire the ability to produce energy through oxidative phosphorylation.  This endosymbiotic theory of eukaryotic evolution also postulates that endosymbiosis resulted in larger eukaryotic genomes, which originated from the partial transfer of mitochondrial genes to the nuclear genome.  On integration into the host genome, mitochondria-derived genes became indistinguishable from the original nuclear genes.Researchers have alternatively proposed that after a prolonged period of symbiosis, there is a possibility of gene transfer from eukaryotes to prokaryotes.  This hypothesis was initially supported when copper/zinc (Cu/Zn) superoxide dismutase (SOD), a metalloprotein confined to the cytosol of eukaryotic cells, was found in Photobacterium leiognathi.  The free-living bacterium P. leiognathi is also a known symbiont of ponyfish, a small fish species native to the Indian and Pacific Oceans.SODs are antioxidant enzymes that serve as the cell's first line of defense against reactive oxygen species (ROS).  ROS produced by the electron transport chain damage proteins by oxidizing amino acid residues and metal ions on prosthetic groups, but can accumulate during times of biochemical and environmental stress.  Superoxide (O₂⁻) radicals, a form of ROS, are sequestered by SODs and converted into less toxic hydrogen peroxide (H₂O₂) and O₂ gas. Adapted from Bannister JV, Parker MW. The presence of a copper/zinc superoxide dismutase in the bacterium Photobacterium leiognathi: a likely case of gene transfer from eukaryotes to prokaryotes. Proc Natl Acad Sci USA. 1985;82(1):149-52. -Investigators used a ribosome profiling technique to measure the level of SOD protein synthesis in P. leiognathi and ponyfish cells during ROS accumulation.  P. leiognathi cells were found to contain greater numbers of ribosome/SOD mRNA complexes than ponyfish cells.  What is the best explanation for this result?

Passage In the central nervous system, myelin produced by oligodendrocytes (glial cells) functions as an insulating sheath surrounding certain nerve fibers.  To identify candidate genes involved in the myelination process, researchers collected oligodendrocytes from zebrafish and purified the messenger RNA (mRNA) transcripts expressed by these glial cells.  The mRNA was converted to complementary DNA (cDNA) by reverse transcriptase, and the cDNA was fluorescently labeled and assessed for hybridization on a microarray.  The expression of mRNA in other zebrafish cells was similarly measured.Transcripts detected in zebrafish oligodendrocytes at levels greater than 3 times those found in other cells were selected as candidate myelination genes.  Some of the detected genes, including plp1a, Sox10, and mbp, were previously known to be specific to oligodendrocytes, validating the procedure.  The cDNA of newly identified candidates was amplified by PCR, and the ends were digested with the restriction enzymes EcoRI and XhoI.  Genes were then ligated into the multiple cloning site (MCS) of the pSKII vector, shown in Figures 1 and 2, which had also been digested by EcoRI and XhoI. Figure 1  Overview of the pSKII vector (plasmid) Figure 2  The pSKII MCS, showing the restriction sites of several restriction enzymesTable 1 shows the functional parameters of several restriction enzymes. To confirm that the candidate genes originated in oligodendrocytes, complementary RNA (cRNA) strands were synthesized from the cloned plasmids and hybridized to fixed zebrafish sections.  One of the genes that localized to oligodendrocytes, known as cldnk, produces a 915 base pair mRNA transcript from two exons.  The cldnk gene is involved in tight junction formation and may be required for myelin sheath development around axons. -How would expression of cloned cldnk differ from expression of the endogenous cldnk gene?

Passage Obesity is a condition that affects over 30% of adults in the United States and is associated with comorbidities such as type 2 diabetes mellitus, heart disease, and cancer.  For individuals with clinically severe obesity, defined as having a body mass index above 40 kg/m², lifestyle interventions may produce insufficient weight reduction.  Weight loss surgery, such as Roux-en-Y gastric bypass (RYGB), has proved to be the most effective clinical intervention for facilitating massive and immediate weight loss in clinically obese patients.In RYGB, the stomach is first divided into a small gastric pouch and a distal gastric remnant, which remains attached to the duodenum.  Next, the jejunum is transected and its lower section is connected to the newly created gastric pouch to form the alimentary limb.  This connection is typically created with surgical staples.  The formation of the alimentary limb allows ingested food to bypass the region composed of the distal gastric remnant, the duodenum, and the upper jejunum; collectively termed the biliopancreatic limb.  Finally, the lumen of the upper jejunum is reconnected to the lumen of the lower jejunum to allow enzymes and compounds to flow freely from the biliopancreatic limb into the alimentary limb. Figure 1  RYGB surgically divides the digestive tract into the alimentary limb and the biliopancreatic limb, which then merge into the common limb.RYGB facilitates weight loss by reducing the functional volume of the stomach and altering intestinal anatomy to induce nutrient malabsorption.  However, the procedure is associated with several complications, such as steatorrhea, or fatty stools, due to disruption of lipid digestion.  Animal studies also suggest that RYGB promotes the growth of weight loss-inducing gut bacteria.  Researchers have found that transplanting gut microbiota of obese postoperative RYGB mice into nonoperated, obese mice lacking gut flora may reproduce the therapeutic effects of RYGB in transplant-recipient mice. Adapted from Bächler T, Le roux CW, Bueter M. How do patients' clinical phenotype and the physiological mechanisms of the operations impact the choice of bariatric procedure?. Clin Exp Gastroenterol. 2016;9:181-9. -A staple line leak is an unanticipated opening of surgical staples at an incision.  In RYGB, a staple line leak at the junction of the biliopancreatic limb and the alimentary limb would cause gastrointestinal tract contents to enter:

Which of the following helps maintain the resting membrane potential of a neuron?Passive transportAdenosine triphosphateMembrane selective permeability

Passage The Bloom syndrome helicase (BLM) transcript is catalytically inactive in Bloom syndrome (BS), a genetic disorder that leads to genomic instability characterized by excessive somatic recombination events.  Research studies have shown that BLM may be involved in DNA synthesis repair of stalled replication forks during replication stress (arrest).  To investigate this further, an experiment was conducted using cell lines derived from a patient with BS.  One set of cells differed only by BLM status:  PSNF5 (BLM⁺, vector with BLM cDNA transfected) and PSNG13 (BLM⁻, empty vector transfected).  Additional mutations, BLM-K695T and BLM-T99A, were studied in stable transfected cells.The nucleoside analogs iododeoxyuridine (IdU) and chlorodeoxyuridine (CldU) were used to label and track genomic regions of active replication.  Cells were initially treated with IdU for 15 minutes.  Subsequently, either 4 mM of hydroxyurea (HU) or 30 µM of aphidicolin (Aph) were used to induce replication stress for 4 hours, after which cells were treated with CldU for 85 minutes to assess replication recovery.  Cells were mounted on microscope slides, lysed to isolate chromosome fibers, and then fixed on the slides.  Sites of IdU and CldU incorporation into the growing DNA strand were then detected by immunostaining the isolated chromosome fibers with analog-specific, fluorescent-coupled antibodies.  Replication activity was visualized via microscopy and measured. Figure 1  (A) Experimental protocol and (B) fluorescent imaging of a microscope slide showing incorporation of IdU (black lines) and CldU (gray lines) into DNA fibers isolated from a group of BLM⁺ and BLM⁻ cells, with single fibers depicted in each row Figure 2  Replication fork recovery in cells expressing BLM⁺, BLM⁻, and the mutation BLM-K695T (antagonistic to wild-type) Figure 3  New sites of replication in cells expressing BLM⁺, BLM⁻, and the mutation BLM-T99A (T99 phosphorylated after replication arrest) Adapted from Davies SL, North PS, Dart A, Lakin ND, Hickson ID. Mol Cell Biol. 2004;24(3):1279-91. -After the 85-minute period, deoxyribonucleoside triphosphates (dNTPs) are added and DNA synthesis is allowed to continue for 20 minutes.  Which of the following processes is most likely to occur during the new replication period?  The structure of CldU is shown below.

Passage The heart is a muscular organ comprising four chambers, two atria and two ventricles, that function synergistically to pump blood through a vast, closed network of blood vessels.  The chambers are separated by membranous muscular barriers known as septa.  Oxygenated blood returning from the lungs via the pulmonary veins fills the left atrium and enters the left ventricle, which then pumps the blood into the systemic arteries to supply other organs in the body.  By contrast, deoxygenated blood returning from systemic veins first enters the right atrium, then the right ventricle, and is ultimately siphoned into the pulmonary arteries leading to the lungs.  The necessary oxygen, nutrient, and waste exchange occurs between the thinnest blood vessels (capillaries) and neighboring tissues of the systemic and pulmonary circuits.Blood flow throughout the circulatory system is dictated by blood (hydrostatic) pressure, vascular resistance (force opposing blood flow through a vessel), and cardiac output (blood volume expelled from the ventricles per unit time).  When blood traverses a vessel, it exerts hydrostatic pressure on the vessel walls, which results in the forced movement of fluid out of the vessel and into the interstitial space.  The circulating plasma proteins cause the osmotic pressure within the vessel to be higher than that of the interstitial fluid.  In turn, osmotic pressure causes fluid to flow from the interstitial space into the blood vessel, opposing hydrostatic pressure.Cardiac output depends in part on heart rate, which is tightly regulated by the sinoatrial (SA) and atrioventricular (AV) nodes, specialized groups of self-depolarizing cells located in the upper right atrial wall and lower interatrial septum, respectively.  Action potentials (APs) generated by SA nodal cells stimulate atrial contraction as they travel to the AV node.  The AV node delays AP transmission to ventricular cells, ensuring ventricular filling is complete prior to heart contraction.A 63-year-old male patient collapsed while exercising and was hospitalized.  The patient presented with an elevated heart rate, low systemic blood pressure, and abnormally low blood oxygen levels.  In addition, x-rays revealed excess fluid in his lungs. -Given the information in the passage, the excess fluid in the patient's lungs is most likely caused by which of the following at the pulmonary sites of gas exchange?

Passage Obstructive respiratory illnesses are characterized by an increased difficulty exhaling much of the air in the lungs.  Asthma and chronic obstructive pulmonary disease (COPD) are two of the most common obstructive respiratory illnesses.  Asthma is due to allergen hypersensitivity and can be identified by temporary airway inflammation, which causes the bronchioles to narrow.  The decreased airway diameter increases the resistance to airflow.  Subsequently, air becomes "trapped" in the lungs, restricting the amount of air that can be moved in each breath.COPD refers to a spectrum of disorders and can be a result of emphysema, the irreversible destruction of pulmonary tissue.  The breakdown of elastic proteins in the lungs results in a loss of pulmonary resiliency and chronic lung hyperinflation.  Because the walls between individual alveoli are broken down, the alveolar sacs that remain are larger.Lung diseases can be diagnosed and monitored using spirometry.  A spirometer measures the flow rate and volume of inhaled and exhaled air.  In one pulmonary test, a patient is asked to exhale forcibly and completely into a spirometer after maximum inhalation.  This test is done to measure the patient's forced expiratory volume in one second (FEV1) and forced vital capacity (FVC), the total amount of air exhaled in a single breath. Figure 1  Forced expiratory spirogram of a healthy individual Adapted from T. Barreiro, "An Approach to Interpreting Spirometry." American Family Physician. ©2004 AAFP. -The inflation of the lungs in normal inspiration involves:Contraction of the diaphragmReduction of intrapleural pressureElevation of the rib cage

Passage The nicotinic acetylcholine receptor (nAChR) is a protein embedded in the muscle fiber membrane that facilitates cell-to-cell communication across the neuromuscular junction.  When acetylcholine (ACh) binds nAChR, electrical depolarization within skeletal muscle fibers is induced and ultimately gives rise to contractile activity.  The nAChR is composed of five protein subunits symmetrically organized around a central ion pore.  Previous studies demonstrated that these subunits may serve as epitopes (antibody binding sites) in autoimmune disease.  By reversibly binding nAChR subunits, anti-nAChr antibodies may temporarily block the attachment site for ACh, resulting in muscle weakness.Researchers harvested muscle samples from mice to investigate the effects of anti-nAChR antibodies on muscle tension, the pulling force transmitted by muscles during contraction.Experiment 1First, skeletal muscle samples (tibialis anterior) of adult male mice were surgically harvested (n = 30), washed, and stripped of connective tissues.  Next, the isolated muscle samples were placed in a 150-mL oxygen-infused, electrolyte-rich bath containing glucose and Ca²⁺ ions that were able to enter the muscle fibers and enable maximal contractile force.  One end of the submerged muscle was anchored in place while the other was attached to an apparatus that measures muscle tension and modifies muscle length.  Using electrodes, action potentials were elicited in the harvested muscle tissue to evaluate contractile function (Figure 1). Figure 1  Action potential elicited from a harvested skeletal muscle sampleExperiment 2The researchers divided the 30 muscle samples into three groups of 10.  Resting muscle tension was measured, and the electrolyte-rich baths of each group were further infused with a 7.5-mL solution containing mouse-specific anti-nAChR antibodies, ACh, or a mixture of both (Table 1).  Post-infusion (peak) muscle tension was measured for 30 minutes, with results displayed in Figure 2.Table 1  Solutions Infused into the Electrolyte-Rich Bath Figure 2  Pre-infusion resting muscle tension and post-infusion peak muscle tension in harvested muscle samples (Note: * indicates p < 0.05.) -To interpret the results of Experiment 2, researchers must assume that:muscle samples were of uniform weight and size.no intermolecular interactions occurred between ACh and anti-nAChR antibodies in Infusion C.co-infusion of ACh and anti-nAChR antibodies was performed following ACh infusion.

The DNA of telomeres can be most accurately described as:

A physician finds that a patient experiences excessive bleeding after sustaining a cut on the arm.  The excessive bleeding is LEAST likely to be caused by:

Passage The Bloom syndrome helicase (BLM) transcript is catalytically inactive in Bloom syndrome (BS), a genetic disorder that leads to genomic instability characterized by excessive somatic recombination events.  Research studies have shown that BLM may be involved in DNA synthesis repair of stalled replication forks during replication stress (arrest).  To investigate this further, an experiment was conducted using cell lines derived from a patient with BS.  One set of cells differed only by BLM status:  PSNF5 (BLM⁺, vector with BLM cDNA transfected) and PSNG13 (BLM⁻, empty vector transfected).  Additional mutations, BLM-K695T and BLM-T99A, were studied in stable transfected cells.The nucleoside analogs iododeoxyuridine (IdU) and chlorodeoxyuridine (CldU) were used to label and track genomic regions of active replication.  Cells were initially treated with IdU for 15 minutes.  Subsequently, either 4 mM of hydroxyurea (HU) or 30 µM of aphidicolin (Aph) were used to induce replication stress for 4 hours, after which cells were treated with CldU for 85 minutes to assess replication recovery.  Cells were mounted on microscope slides, lysed to isolate chromosome fibers, and then fixed on the slides.  Sites of IdU and CldU incorporation into the growing DNA strand were then detected by immunostaining the isolated chromosome fibers with analog-specific, fluorescent-coupled antibodies.  Replication activity was visualized via microscopy and measured. Figure 1  (A) Experimental protocol and (B) fluorescent imaging of a microscope slide showing incorporation of IdU (black lines) and CldU (gray lines) into DNA fibers isolated from a group of BLM⁺ and BLM⁻ cells, with single fibers depicted in each row Figure 2  Replication fork recovery in cells expressing BLM⁺, BLM⁻, and the mutation BLM-K695T (antagonistic to wild-type) Figure 3  New sites of replication in cells expressing BLM⁺, BLM⁻, and the mutation BLM-T99A (T99 phosphorylated after replication arrest) Adapted from Davies SL, North PS, Dart A, Lakin ND, Hickson ID. Mol Cell Biol. 2004;24(3):1279-91. -What is the most accurate description of Aph-treated cells compared to wild-type cells treated with the same agent?

Passage Rheumatoid arthritis (RA) is an autoimmune disease that causes chronic synovial (joint) inflammation.  RA immunopathogenesis involves LFA-1, a surface receptor found on leukocytes that is composed of an α-chain (CD11a) and a β-chain (CD18).  The binding of LFA-1 to ICAM-1, an adhesion protein on the surface of B cells and endothelial cells, stimulates the migration of leukocytes to specific areas of inflammation.  After successful LFA-1/ICAM-1 binding, CD18 is released into the plasma and synovial fluid (SF), where it exists in soluble form (sCD18).  The levels of sCD18 in SF can be used as a marker for inflammation in RA.CD18 release has been shown to increase in response to tumor necrosis factor (TNF), a pro-inflammatory cytokine involved in the inflammatory response of RA.  In patients with newly diagnosed and untreated RA (eRA), leukocytes produce adequate quantities of LFA-1 and plasma sCD18 levels are unchanged.  However, over time there is dysregulation of LFA-1 synthesis and an increase in competition for ICAM-1 by other ligands, leading to a reduction in CD18 shedding.  As a result, sCD18 is decreased in patients with chronic RA (cRA).Experiment 1An immunofluorescence assay was used to measure plasma sCD18 levels in patients with eRA and cRA, and in age- and gender-matched healthy controls (HC). Figure 1  Baseline sCD18 measurements (Note: 50% of data points are below the median value; median = line inside the box.)Experiment 2Clinical assessments were performed in eRA patients to establish a baseline disease status.  eRA patients were assigned DAS28CRP scores measured by RA-specific laboratory values.  This was followed by treatment with anti-inflammatory drugs.  DAS28CRP scores were calculated at 3-month intervals for 12 months, with lower scores indicating decreased RA severity (Figure 2). Figure 2  DAS28CRP scores in eRA patientsExperiment 3Heterogeneous populations of mononuclear cells were cultured from the synovial fluid (SFMC) and peripheral blood (PBMC) of eRA patients during an acute exacerbation of their symptoms.  sCD18 levels were measured in SFMCs and PBMCs that were untreated, treated with TNF, or treated with monoclonal antibodies targeting TNF (anti-TNF). Adapted from Kragstrup TW, Jalilian B, Keller KK, et al. Changes in Soluble CD18 in Murine Autoimmune Arthritis and Rheumatoid Arthritis Reflect Disease Establishment and Treatment Response. PLoS ONE. 2016;11(2):e0148486. -Which statement about RA is best supported by the information in the passage?

Passage Saliva, a secreted fluid with multiple physiologic functions, is composed of water (~99%), proteins, and electrolytes.  Together, these components facilitate nutrient consumption, respiration, and communication.  For example, saliva aids in digestion by serving as a lubricant during mastication and by initiating carbohydrate and fat metabolism.  In addition, the calcium and phosphate present in saliva promote tooth remineralization, and antimicrobial and chemical buffering agents give saliva protective properties.The gustatory-salivary reflex, the secretion of saliva from the salivary glands in response to gustation (taste), is primarily mediated by the parasympathetic division of the nervous system.  In general, parasympathetic pathways originate from the brain stem or the sacral spinal cord.  Fibers originating from nuclei in these regions synapse onto ganglia that lie in close proximity to the target organ.  Both preganglionic neurons (those originating from the nuclei) and postganglionic neurons (those originating from the ganglia) release acetylcholine (ACh).In the gustatory-salivary reflex, activation of taste receptors in the tongue sends impulses to the superior and inferior salivatory nuclei in the brainstem.  Local processing within these nuclei activates the parasympathetic pathways that innervate the salivary glands.  Ultimately, ACh released from postganglionic neurons acts on the muscarinic acetylcholine receptors (mAChRs) on the secretory cells of the salivary gland to stimulate salivation (Figure 1). Figure 1  Parasympathetic regulation of salivary secretionSympathetic stimulation of the salivary glands also promotes salivation but to a lesser degree than parasympathetic stimulation.  This may contribute to what is often perceived to be decreased salivation when the sympathetic division is more active than its counterpart.  Unlike parasympathetic pathways, sympathetic pathways originate in the thoracic and lumbar spinal segments, and the ganglia lie in close proximity to the spinal cord.  Although preganglionic neurons of the sympathetic nervous system release ACh, the postganglionic neurons often release norepinephrine. -The gustatory-salivary reflex pathway is part of which of the following branches of the nervous system?PeripheralSomaticAutonomic

Passage Lipopolysaccharides (LPS) found in gram-negative bacterial cells can elicit a lethal degree of adjuvanticity, or immune activation, in LPS-responsive host organisms.  In mice, responses may be modulated by abnormalities in the toll-like receptor (TLR) encoded by the Tlr4 gene closely linked to coat color on Chromosome 4.  To investigate the effect of a particular Tlr4 mutation on LPS response, three experiments were performed following a cross between fully responsive He-N mice [tlr4(+)/tlr4(+)] and He-J mice homozygous for the mutation [tlr4(d)/tlr4(d)].Experiment 1Researchers isolated spleen cells for analysis of protein expression and LPS response.  In cells isolated from F1 mice, both wild-type and mutant TLRs were distinguishable by protein electrophoresis.  Following this first analysis, spleen cells from each cohort were exposed to 2 × 10⁵ SRC, an antigen formed by sheep erythrocytes.  Cultures were then incubated in the presence or absence of Ph-LPS, with LPS extracted from Escherichia coli K345 cells using a phenol-water mixture.  Titers of anti-SRC antibody were recorded on incubation day 5, with the results shown in Table 1.Table 1  Mean anti-SRC titers with standard error Experiment 2Serum samples were assayed for interferon production 2 hours post LPS injection.  Interferon was quantified using a plaque reduction assay on mouse cells challenged with vesicular stomatitis virus. Figure 1  Average serum interferon levels in mice cohortsExperiment 3Mice were injected with varying doses of Ph-LPS and monitored for 4 days.  The lethal dose (LD₅₀) was defined as the minimum dose causing death in 50% of injected mice.  Compared to He-N mice, results reflected a 10-fold increase in LD₅₀ for F1 mice and a 100-fold increase in LD₅₀ for He-J mice. -If researchers repeated the analyses using F2 generation mice, spleen cell cultures exposed to SRC plus 1.0 μg Ph-LPS would be expected to have anti-SRC titers of:16001200800

Passage Trypanosoma brucei is a eukaryotic parasite that causes sleeping sickness in Sub-Saharan Africa.  The cell cycle of T. brucei differs from that of higher eukaryotes.  For example, the chromatin does not condense during mitosis, and the nuclear envelope remains intact.  In addition, the nucleus divides before cell division, and the cell divides along the longitudinal axis (Figure 1). Figure 1  Mitosis in T. bruceiA culture of T. brucei was arrested in S phase of the cell cycle by overnight incubation with the reversible inhibitor hydroxyurea.  The cells were then washed, and their progression through the cell cycle was monitored by flow cytometry and microscopy.  A subset of the culture was first exposed to the drug VX-680, which arrests cells upon completion of metaphase.  The results are shown in Figure 2. Figure 2  Percentages of a population of T. brucei cells in each phase of the cell cycle, measured at different time points in the (A) absence and (B) presence of VX-680 Adapted from Li Z, Umeyama T, Wang CC. The Aurora Kinase in Trypanosoma brucei plays distinctive roles in metaphase-anaphase transition and cytokinetic initiation. PLoS Pathog. 2009;5(9):e1000575. -Why were T. brucei cells incubated with hydroxyurea overnight and washed afterward?

Cells of the innate immune system and cells of the adaptive immune system differ in that only the latter:

Passage The fruit fly Drosophila melanogaster is frequently used as a model system to investigate neurological disorders.  The genes Abl, Enabled (Ena), and receptor protein tyrosine phosphatase (PTP) are involved in axonal guidance during neuronal development and are all believed to operate in the same pathway.  To investigate interactions between PTP and either Ena or Abl, fly lines were developed in which mutations of each gene were easily identifiable by eye characteristics.  In flies that would normally have white eyes, a dominant mutation that inactivates PTP results in red and white eyes; a dominant Ena mutation yields orange eyes (or red and orange if the PTP and Ena mutations are both present); and a dominant Abl mutation yields elongated eyes.  Using these fly lines, the following experiments were conducted.Experiment 1Females heterozygous for PTP (eePp) were crossed with males heterozygous for Ena (Eepp).  F1 generation males and females, each with red and orange eyes, were then selected and crossed with each other to produce an F2 generation.  Flies with two mutant Ena alleles (EE), as well as those with two mutant PTP alleles and at least one mutant Ena allele (EePP), did not survive past the larval stage.Experiment 2PTP and Abl are located on the same chromosome.  Females with the PTP mutation on one copy of the chromosome and the Abl mutation on the other copy were crossed with wild-type males.  In Drosophila, recombination occurs only in female gametes.  Progeny with parental and recombinant genotypes are shown in Table 1.  F1 males with red and white elongated eyes were then crossed with homozygous PTP mutant females.  This produced an F2 generation in which 50% of both male and female progeny had elongated eyes.Table 1  Phenotypes of 500 F1 Flies Like humans, male flies have a single X chromosome, whereas females have two X chromosomes.  Unlike humans, female Drosophila can store sperm and fertilize multiple eggs over the course of several days after a single mating.  Researchers ensured that females were virgins prior to setting up each cross to avoid contamination. Adapted from Song JK, Giniger E, Desai CJ. The receptor protein tyrosine phosphatase PTP69D antagonizes Abl tyrosine kinase to guide axons in Drosophila. Mech Dev. 2008;125(3-4):247-56. -In Experiment 1, approximately what fraction of the F2 generation is expected to die during the larval stage?