Pathology Image Of the Month: Rapidly Progressive Hemorrhagic Cellulitis of Bilateral Lower Extremities with Subsequent Septic Shock and Death.

A 51-year-old man presented to a community based emergency department with bilateral lower extremity swelling that began four days prior and that had evolved into recent blister formation on the left lower extremity. Medical history was significant only for hypertension and a recent self-described episode of "food poisoning" five days earlier characterized by diarrhea, nausea, and vomiting that quickly resolved. Physical exam revealed marked bilateral lower extremity edema and an ecchymotic rash below the knee. In addition to the rash, there were large flaccid bullae on the left leg, mostly intact but some notable for draining of scanty serosanguinous fluid. The patient was tachycardic with a rate of 114 bpm and initial labs showed thrombocytopenia (platelets 56 x 103/uL [140-440 x 103/uL]), hypoglycemia (15mg/dl [70-105mg/dl]), an elevated creatinine (2.7mg/dL [0.7- 1.25mg/dL]), and aspartate aminotransferase (AST 156U/L [5- 34U/L]). Two sets of blood cultures were drawn, broad spectrum antibiotics including doxycycline were empirically initiated and then he was subsequently transported to a tertiary care hospital for escalation of care. Within hours of presentation to the tertiary care facility, the rash appeared progressively hemorrhagic and bullous, lactic acidosis and coagulopathy developed and hemodynamic instability and septic shock necessitated endotracheal intubation and vasopressors. He was taken to the operating room for skin debridement but was emergently converted to bilateral above the knee lower extremity amputations due to the extent of the soft tissue necrosis. The patient remained intubated and in critical condition following surgery and the ecchymotic rash reappeared at the amputation sites. A newly developed ecchymotic rash with bullae formation was noted on the right upper extremity forearm. At that time, the clinicians were notified that four out of four blood culture bottles from admission were rapidly growing a microorganism. The family elected for withdrawal of care, and the patient died approximately 72 hours following presentation. A full and unrestricted autopsy was authorized by the Coroner's Office.

Pathology Image Of The Month: Recurrent Urinary Tract Infections In a 48-year-Old Female Hyperintensities in the Globus Pallidi.

A 48-year-old female presented to her physician complaining of intermittent lower abdominal pain radiating to the right lower back. At the time of presentation, she was afebrile and denied any urinary symptoms such as dysuria, frequency or urgency. Physical exam was unremarkable, other than obesity, and her abdominal exam was noncontributory. Medical history, however, was notable for recurrent, Proteus mirabilis culture-proven urinary tract infections requiring antibiotic treatment for the prior three years along with hypertension, uterine fibroids post hysterectomy, diabetes and asthma. Complete blood count was within normal limits, and urine dipstick showed 1+ blood, 1+ protein and 2+ leukocytes. Full renal function labs, urine cytology, radioisotope renography and abdominopelvic CT scanning with contrast were all ordered and a laparoscopic right nephrectomy was recommended based on the results. The bisected right kidney is shown below in Figure 1. She was discharged home on post-operative day one and her follow up urology appointments indicate resolution of both her urinary tract infections and her abdominal pain.

PATHOLOGY IMAGE OF THE MONTH. Autopsy Findings in an Adult with Down Syndrome.

Emergency medical responders were activated to the home of a 59-year-old African-American male in distress and with known Down syndrome complicated by Alzheimer's disease. He was found to be unresponsive and subsequently became pulseless. Advanced cardiac life support protocols were initiated and continued for two hours in the emergency department. Due to family request, efforts were eventually ceased and the patient was declared dead. Full, unrestricted autopsy examination was conducted under the coroner's authorization. The cause of death was determined to be a pulmonary thromboembolus in the main pulmonary artery with extension into the bilateral pulmonary arteries. Additional external findings included alopecia universalis, penoscrotal hypospadias, ostium secundum type of atrial septal defect, right ventricular cardiac dilatation, diffuse cerebral atrophy, facial features compatible with Down syndrome, and generalized patches of skin depigmentation over the hands as seen in Figure 1 but also over the feet, lips, areola, and trunk. Microscopic findings included features of pulmonary hypertension. A microscopic image from a section of the thyroid is seen in Figure 2.

Effect of protein binding on ultrafast DNA dynamics: characterization of a DNA:APE1 complex.

Synthetic oligonucleotides with a fluorescent coumarin group replacing a basepair have been used in recent time-resolved Stokes-shift experiments to measure DNA dynamics on the femtosecond to nanosecond timescales. Here, we show that the APE1 endonuclease cleaves such a modified oligonucleotide at the abasic site opposite the coumarin with only a fourfold reduction in rate. In addition, a noncatalytic mutant (D210N) binds tightly to the same oligonucleotide, albeit with an 85-fold reduction in binding constant relative to a native oligonucleotide containing a guanine opposite the abasic site. Thus, the modified oligonucleotide retains substantial biological activity and serves as a useful model of native DNA. In the complex of the coumarin-containing oligonucleotide and the noncatalytic APE1, the dye's absorption spectrum is shifted relative to its spectrum in either water or within the unbound oligonucleotide. Thus the dye occupies a site within the DNA:protein complex. This result is consistent with modeling, which shows that the complex accommodates coumarin at the site of the orphaned base with little distortion of the native structure. Stokes-shift measurements of the complex show surprisingly little change in the dynamics within the 40 ps-40 ns time range.

Determination of apoptosis, uracil incorporation, DNA strand breaks, and sister chromatid exchanges under conditions of thymidylate deprivation in a model of BER deficiency.

Thymidylate synthase (TS) is an important target of several chemotherapeutic agents. During TS inhibition, dTTP levels decrease with a subsequent increase in dUTP. Uracil incorporated into the genome is removed by base excision repair (BER). BER has been hypothesized to play a role in the response to thymidylate deprivation, despite a lack of direct evidence. We previously found that beta-pol null murine fibroblasts were approximately six-fold more resistant than wild-type cells to raltitrexed, a folate-based inhibitor specific for TS. In this study, a number of endpoints were determined to understand the influence of BER and beta-pol during raltitrexed treatment. Raltitrexed induced apoptosis in wild-type cells to a greater extent than in beta-pol null cells. A PARP inhibitor decreased the sensitivity to raltitrexed, although the extent was not different between wild-type and beta-pol null cells. No evidence was seen for extensive strand break formation that preceded apoptosis, although raltitrexed induced more sister chromatid exchanges in wild-type cells. Increased levels of uracil in DNA were detected following treatment in wild-type and beta-pol null cells. However, uracil levels were only approximately two-fold higher in DNA from treated cells compared to untreated. Uracil DNA glycosylase activity was slightly higher in beta-pol null cells, although not sufficiently different to explain the difference in sensitivity to raltitrexed. Taken together, the data suggest that the sensitivity of the wild-type cells to raltitrexed is not associated with activation of PARP-1 dependent BER, extensive uracil incorporation into DNA and persistent strand breaks, but rather with changes suggestive of DNA recombination.

Effects of substrate specificity on initiating the base excision repair of N-methylpurines by variant human 3-methyladenine DNA glycosylases.

The human 3-methyladenine (AAG, ANPG, MPG) DNA glycosylase excises alkylated purines from DNA. In previous studies, we determined the importance of an active site amino acid (asparagine 169) in the recognition of substrates by AAG. In this study, we characterize the consequences of expressing the AAG variants bearing amino acid substitutions at position 169 in Saccharomyces cerevisiae that lack endogenous 3-methyladenine DNA glycosylase. Survival, mutation induction, and DNA double strand break formation were determined in response to methyl methanesulfonate. The ability of purified wild-type and AAG variants to remove 3-methyladenine and 7-methylguanine, the two most abundant adducts produced by methyl methanesulfonate, was also determined. The N169D AAG variant displayed a approximately 100-fold lower activity for 3-methyladenine as compared to wild-type and did not detectably remove 7-methylguanine. When expressed in S. cerevisiae, the N169D variant provided better protection against methyl methanesulfonate toxicity than wild-type. Fewer strand breaks in vivo were also seen in the presence of the N169D variant following MMS exposure. In contrast, the N169A and N169S AAG variants displayed approximately 30-fold lower activity for 3-methyladenine and 7-methylguanine. Expression of the N169A and N169S AAG variants in S. cerevisiae during methyl methanesulfonate exposure resulted in greater sensitivity, greater mutation induction following MMS exposure, and more strand breaks in vivo. Strand breaks seen in S. cerevisiae that express wild-type AAG or the N169 variants were resolved to varying extents during recovery. In contrast, strand breaks formed in S. cerevisiae that expressed a catalytically inactive AAG variant were not resolved during the recovery times examined. Taken together, the results provide evidence that 3-methyladenine adducts not repaired by base excision repair cause double strand breaks that are not rapidly resolved. Evidence is also provided that the BER intermediates resulting from excision of 7-methylguanine by wild-type AAG contributes to the mutagenicity and cytotoxicity of alkylating agents.

Oxanine DNA glycosylase activity from Mammalian alkyladenine glycosylase.

Oxanine (Oxa) is a deaminated base lesion derived from guanine in which the N(1)-nitrogen is substituted by oxygen. This work reports the mutagenicity of oxanine as well as oxanine DNA glycosylase (ODG) activities in mammalian systems. Using human DNA polymerase beta, deoxyoxanosine triphosphate is only incorporated opposite cytosine (Cyt). When an oxanine base is in a DNA template, Cyt is efficiently incorporated opposite the template oxanine; however, adenine and thymine are also incorporated opposite Oxa with an efficiency approximately 80% of a Cyt/Oxa (C/O) base pair. Guanine is incorporated opposite Oxa with the least efficiency, 16% compared with cytosine. ODG activity was detected in several mammalian cell extracts. Among the known human DNA glycosylases tested, human alkyladenine glycosylase (AAG) shows ODG activity, whereas hOGG1, hNEIL1, or hNEIL2 did not. ODG activity was detected in spleen cell extracts of wild type age-matched mice, but little activity was observed in that of Aag knock-out mice, confirming that the ODG activity is intrinsic to AAG. Human AAG can excise Oxa from all four Oxa-containing double-stranded base pairs, Cyt/Oxa, Thy/Oxa, Ade/Oxa, and Gua/Oxa, with no preference to base pairing. Surprisingly, AAG can remove Oxa from single-stranded Oxa-containing DNA as well. Indeed, AAG can also remove 1,N(6)-ethenoadenine from single-stranded DNA. This study extends the deaminated base glycosylase activities of AAG to oxanine; thus, AAG is a mammalian enzyme that can act on all three purine deamination bases, hypoxanthine, xanthine, and oxanine.

Active-site clashes prevent the human 3-methyladenine DNA glycosylase from improperly removing bases.

The human 3-methyladenine DNA glycosylase (AAG, MPG) removes a diverse array of damaged purines via a nucleotide-flipping mechanism. In the crystal structure of AAG bound to DNA containing 1,N(6) ethenoadenine, an asparagine (N169) occupies the active-site floor, in close proximity to the C-2 position of the flipped-out 1,N(6) ethenoadenine. We engineered site-specific AAG mutants to determine whether N169 prevents normal bases from mistakenly entering the active site. Substituting alanine or serine resulted in mutants that excised substrates at a faster rate than wild-type. Furthermore, these mutants acquired the ability to excise normal guanine within mispairs but not opposite cytosine. The results suggest that AAG can recognize helical deformations, such as mispairs. However, the active site then prevents the mistaken excision of bases, which prevents AAG from acquiring a mutator activity.