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Human reticulocyte 15-lipoxygenase-1 (15-hLO-1) and human platelet 12-lipoxygenase (12-hLO) have been implicated in a number of diseases, with differences in their relative activity potentially playing a central role. In the current paper, we characterize the catalytic mechanism of these two enzymes with arachidonic acid (AA) as the substrate. Using variable-temperature kinetic isotope effects (KIE) and solvent isotope effects (SIE), we demonstrate that both kcat/Km and kcat for 15-hLO-1 and 12-hLO involve multiple rate-limiting steps that include a solvent dependent step and hydrogen atom abstraction. Nevertheless, an unexpectedly low kcat/Km KIE was determined for 15-hLO-1 (KIE = 8), which increases to well above semi-classical predictions (KIE = 18) upon the addition of the allosteric effector molecule, 12-hydroxyeicosatetraenoic acid (12-HETE), indicating a tunneling mechanism. Furthermore, the addition of 12-HETE lowers the observed kcat/Km SIE from 2.2 to 1.4, indicating that the rate-limiting contribution from solvent rearrangement in the reaction mechanism of 15-hLO-1 has decreased, with a concomitant increase in the C-H abstraction contribution. Finally, the allosteric binding of 12-HETE to 15-hLO-1 decreases the Km(O2) for AA, but increases the Km(O2) for LA, such that the Km(O2) values become similar for both substrates (∼20 μM). Considering that the oxygen concentration in cancerous tissue can be below 5 μM, this result may have cellular implications with respect to the substrate specificity of 15-hLO-1.

Lipoxygenases (LOs) catalyze lipid peroxidation and have been implicated in a number of human diseases connected to oxidative stress and inflammation. These enzymes have also attracted considerable attention due to large kinetic isotope effects (30−80) for the rate-limiting hydrogen abstraction step with linoleic acid (LA) as substrate. Herein, we report kinetic isotope effects (KIEs) in the reactions of three human LOs (platelet 12-hLO, reticulocyte 15-hLO-1, and epithelial 15-hLO-2) with arachidonic acid (AA). Surprisingly, the observed KIEs with AA were much smaller than the previously reported values with LA. Investigation into the origins for the smaller KIEs led to the discovery of isotope sensitive branching of the reaction pathways. Product distribution analysis demonstrated an inversion in the regioselectivity of 15-hLO-1, with hydrogen abstraction from C13 being the major pathway with unlabeled AA, but abstraction from C10 predominating when the methylene group at position 13 was deuterated. Smaller but clear changes in regioselectivity were also observed for 12-hLO and 15-hLO-2. Keywordsisotope sensitive branching; lipoxygenase; arachidonic acid Lipoxygenases (LOs) are non-heme iron proteins that catalyze the hydrogen atom abstraction from a bisallylic position in polyunsaturated fatty acids and the subsequent addition of molecular oxygen to the resulting substrate radical to generate hydroperoxide products (1). Mammalian LOs catalyze key steps in the conversion of arachidonic acid (AA), 1 a C20 tetraunsaturated fatty acid (20:4, ω−6), to lipoxins and leukotrienes (2). They have been implicated in a number of pathologies including cancer (3,4), atherosclerosis (5) and Alzheimer's disease (6,7). In plants, LOs convert linoleic acid (LA), a C18 bisunsaturated fatty acid (18:2, ω−6), into 13-hydroperoxyoctadecadienoic acid (13-HPODE), a precursor for † This work was supported by the National Institutes of Health (GM44911, WAV and GM56062, TRH) and American Heart Association pre-doctoral fellowships (0615604Z, CJ and 0310006Z, CMM).vddonk@uiuc.edu. tholman@chemistry.ucsc.edu. Supporting information Synthetic procedures for 10,10,13,13-d 4 arachidonic acid and HPLC traces displaying product distributions of the human lipoxygenase reactions are available free of charge via the internet at http://pubs.acs.org. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2009 July 8. Published in final edited form as:Biochemistry. jasmonates and aldehydes involved in signaling, germination, and senescence (8). In mammals, the isozymes are named according to the position of AA that reacts with molecular oxygen (Figure 1). Several human LOs (5-, 12-, and 15-hLOs) have thus far been identified (9), with this study focusing on the latter two groups. Two isozymes of 15-hLO have been characterized in humans and are called 15-hLO-1 (also termed 12/15-hLO) (10,11) and 15-hLO-2 (12). The former exhibits a product selectivity of 15-HPETE over 12-HPETE of about a 9:1 (13), whereas the lat...

Transfusion‐transmitted infection risk remains an enduring challenge to blood safety in Africa. A high background incidence and prevalence of the major transfusion‐transmitted infections (TTIs), dependence on high‐risk donors to meet demand, suboptimal testing and quality assurance collectively contribute to the increased risk. With few exceptions, donor testing is confined to serological evaluation of human immunodeficiency virus (HIV), hepatitis B and C (HBV and HCV) and syphilis. Barriers to implementation of broader molecular methods include cost, limited infrastructure and lack of technical expertise. Pathogen reduction (PR), a term used to describe a variety of methods (e.g. solvent detergent treatment or photochemical activation) that may be applied to blood following collection, offers the means to diminish the infectious potential of multiple pathogens simultaneously. This is effective against different classes of pathogen, including the major TTIs where laboratory screening is already implemented (e.g. HIV, HBV and HCV) as well pathogens that are widely endemic yet remain unaddressed (e.g. malaria, bacterial contamination). We sought to review the available and emerging PR techniques and their potential application to resource‐constrained parts of Africa, focusing on the advantages and disadvantages of such technologies. PR has been slow to be adopted even in high‐income countries, primarily given the high costs of use. Logistical considerations, particularly in low‐resourced parts of Africa, also raise concerns about practicality. Nonetheless, PR offers a rational, innovative strategy to contend with TTIs; technologies in development may well present a viable complement or even alternative to targeted screening in the future.

Objectives To improve understanding of transition from viral infection to viral clearance, and antibody response in pediatric patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Study design This retrospective analysis of children tested for SARS-CoV-2 by reverse transcription (RT) polymerase chain reaction (PCR) and immunoglobulin G antibody at a quaternary-care, free-standing pediatric hospital between March 13, 2020, and June 21, 2020, included 6369 patients who underwent PCR testing and 215 patients who underwent antibody testing. During the initial study period, testing focused primarily on symptomatic children; the later study period included asymptomatic patients who underwent testing as preadmission or preprocedural screening. We report the proportion of positive and negative tests, time to viral clearance, and time to seropositivity. Results The rate of positivity varied over time due to viral circulation in the community and transition from targeted testing of symptomatic patients to more universal screening of hospitalized patients. Median duration of viral shedding (RT-PCR positivity) was 19.5 days and time from RT-PCR positivity to negativity was 25 days. Of note, patients aged 6 through 15 years demonstrated a longer time of RT-PCR positivity to negativity, compared with patients aged 16 through 22 years (median 32 vs 18 days, P = .015). Median time to seropositivity, by chemiluminescent testing, from RT-PCR positivity was 18 days, whereas median time to reach adequate levels of neutralizing antibodies (defined as comparable with 160 titer by plaque reduction neutralization testing) was 36 days. Conclusions The majority of patients demonstrated a prolonged period of viral shedding after infection with SARS CoV-2. It is unknown whether this correlates with persistent infectivity. Only 17 of 33 patients demonstrated adequate neutralizing antibodies during the time frame of specimen collection. It remains unknown whether immunoglobulin G antibody against spike structured proteins correlates with immunity, and how long antibodies and potential protection persist.

We present the case of a 46-year-old woman with no significant past medical history who developed left mid-thigh pain and fullness. Imaging demonstrated a mineralized soft-tissue mass, which increased in size during a year of monitoring, but retained a circumscribed appearance. The mass was located in the medial soft tissues of the thigh, separate from the bone on imaging studies, and this finding was confirmed during excision. The mass showed gross and microscopic features of an aneurysmal bone cyst. This diagnosis was supported by cytogenetic analysis revealing a t(17;17)(p13;q21) translocation corresponding to the USP6 and COL1A1 loci. Soft-tissue aneurysmal bone cyst is a rare entity, with fewer than 25 reports in the literature. Limited cytogenetic information about these tumors is available. To our knowledge, the USP6 and COL1A1 rearrangement has only previously been described in a pediatric soft-tissue aneurysmal bone cyst. We also discuss the differential diagnosis of ossifying soft-tissue lesions.

The reaction of soybean lipoxygenase-1 with linoleic acid has been extensively studied and displays very large kinetic isotope effects. In this work, substrate and solvent kinetic isotope effects as well as the viscosity dependence of the oxidation of arachidonic acid were investigated. The hydrogen atom abstraction step was rate-determining at all temperatures, but was partially masked by a viscosity-dependent step at ambient temperatures. The observed KIEs on k cat were large (~100 at 25°C).Lipoxygenases are non-heme iron-dependent proteins that catalyze the oxidation of polyunsaturated fatty acids to hydroperoxides. 1 These enzymes occur in plants and animals, and have also been detected in certain bacteria. 2,3 Lipoxygenase products are further elaborated by downstream enzymes to give a variety of physiological regulators and pain mediators. In plants, lipoxygenases convert fatty acids into jasmonates and aldehydes, which are involved in signaling, germination and senescence. 4 Soybean lipoxygenase-1 (sLO-1) exhibits high structural similarity with mammalian lipoxygenases despite only sharing 25% sequence identity. 5 sLO-1 acts on polyunsaturated fatty acids in which a 1,4-diene unit is located six carbons away from the methyl terminus (ω-6 fatty acids). Its natural substrate is linoleic acid (LA), a C18 bisunsaturated fatty acid, which it converts into 13-hydroperoxyoctadienoic acid (13-HPODE). sLO-1 can also catalyze the oxidation of arachidonic acid (AA), a C20 tetraunsaturated fatty acid found in animals, to 15-hydroperoxyeicosatetraenoic acid (15-HPETE).Lipoxygenases abstract a hydrogen atom from the substrate to form a radical, which then reacts with molecular oxygen. 6,7 In resting lipoxygenase, the iron is in the ferrous form and the enzyme is inactive. 8 Before catalysis can occur, the iron must be converted to the active ferric hydroxide form by autooxidized compounds. A subsequent proton-coupled electron transfer from the substrate to the ferric hydroxide forms an intermediate radical and a ferrous species (Figure 1). 9,10 After stereoselective reaction of the substrate radical with molecular oxygen, the peroxyl radical oxidizes the iron back to the active ferric state and the peroxide product is released from the enzyme.Lipoxygenases have drawn considerable attention due to the large kinetic isotope effects (KIEs) exhibited in reactions with LA. 11-14 The observed values of 50-100 for k cat are much larger than the semi-classical limit. Quantum chemical tunneling in the rate-determining hydrogen atom transfer step, mediated by protein dynamics, has been proposed to account for these largePublisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered whic...

BACKGROUND Alloantibodies against more than 50 non‐ABO blood group antigens have been implicated in hemolytic disease of the fetus and newborn (HDFN) and are expected to wane within weeks after delivery. Persistent anemia leads to the hypothesis of continued exposure to red blood cell (RBC) alloantibodies via breast milk, which has been shown in a murine model and suggested in rare case reports. CASE REPORT We report three cases of prolonged HDFN in two neonates with anti‐D HDFN and one with anti‐Jka HDFN. Patient 1 demonstrated 4+ anti‐D serologic testing beyond 2 months; therefore, antibody testing was performed on maternal breast milk. METHODS Maternal serum samples were tested for the presence of unexpected antibodies using standard Ortho gel card and 37 °C 60 minutes with anti‐human globulin (AHG) tube saline methods. Antibody titrations were performed using the standard 37 °C 60 minutes to AHG tube saline method. Fresh breast milk samples were tested using the standard 37 °C 60 minutes to AHG tube saline method for both unexpected antibodies and titration study. Fresh breast milk from an O‐positive, antibody‐negative donor was used as control for any reactivity that may have been due to milk solids or proteins alone. RESULTS Using a known methodology applied in a novel way to test breast milk for RBC alloantibodies, antibodies against fetal RBCs were identified in the maternal breast milk of three patients. CONCLUSION Maternal RBC alloantibodies are present in breast milk and may be clinically significant in patients with prolonged recovery from HDFN.