Caprylate/chromatography process to produce highly purified tetanus immune globulin from human plasma.

While tetanus toxoid vaccination has reduced the incidence of tetanus in the developed world, this disease remains a substantial health problem in developing nations. Tetanus immune globulin (TIG) is used along with vaccination for prevention of infection after major or contaminated wounds if vaccination status cannot be verified or for active tetanus infection. These studies describe the characterisation of a TIG produced by a caprylate/chromatography process. The TIG potency and presence of plasma protein impurities were analysed at early/late steps in the manufacturing process by chromatography, immunoassay, coagulation and potency tests. The caprylate/chromatography process has been previously shown to effectively eliminate or inactivate potentially transmissible agents from plasma-derived products. In this study, the caprylate/chromatography process was shown to effectively concentrate TIG activity and efficiently remove pro-coagulation factors, naturally present in plasma. This TIG drug product builds on the long-term evidence of the safety and efficacy of TIG by providing a product with higher purity and low pro-coagulant protein impurities.

Application of a caprylate/chromatography purification process for production of a high potency rabies immune globulin from pooled human plasma.

BACKGROUND: Human rabies immunoglobulin (RIG) is an integral part of post-exposure prophylactic treatment of rabies (along with rabies vaccination). Infiltration of most, if not all, of the RIG dose at the wound site is recommended. RIG produced by a caprylate/chromatography manufacturing process (RIG-C; HyperRAB) increased the potency and purity of this product over the existing licensed RIG from a solvent/detergent process (RIG-S/D; HyperRAB-S/D). METHODS: A series of studies were conducted to characterize the content and purity of RIG-C. A single-dose pharmacokinetic study in rabbits was performed to compare intramuscular (IM) immunoglobulin products manufactured by two different purification processes, solvent/detergent (IGIM-S/D) and caprylate/chromatography (IGIM-C). RESULTS: RIG-C was found to be a highly purified IgG formulation with high monomer content and formulated with twice the anti-rabies potency of RIG-S/D while maintaining the same overall protein concentration. RIG-C facilitates IM administration at the wound site by halving the injection volume. The new caprylate/chromatography process eliminated detectible levels of pro-coagulant impurities and IgA that were carried through in the prior S/D process. These impurities have been associated with thrombotic complications and allergic reactions in susceptible patients. After single dose administration, IGIM-C was pharmacokinetically equivalent to IGIM-S/D in rabbits. CONCLUSION: RIG-C is a more potent RIG formulation with less impurities yielding a safer and more convenient product with similar pharmacokinetic profile.

Internal Carotid Artery Agenesis in a Trauma Patient with Ipsilateral Neck GSWs.

A 38-year-old man presented to the emergency room in the trauma bay for multiple ballistic injuries to the right neck. He was hemodynamically stable, protecting his airway, and neurologically intact. Computed tomography angiography (CTA) revealed absent filling the right internal carotid artery from its origin to the circle of Willis, which was intact, as well as absent petrous carotid canal on the right. The patient was diagnosed with right internal carotid artery (ICA) agenesis and discharged in several days. This report demonstrates the importance of an in-depth knowledge of vascular embryology and anatomy. The patient has agreed to have images and case details published.

Production of anti-SARS-CoV-2 hyperimmune globulin from convalescent plasma.

BACKGROUND: In late 2019, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus emerged in China and quickly spread into a worldwide pandemic. Prior to the development of specific drug therapies or a vaccine, more immediately available treatments were sought including convalescent plasma. A potential improvement from convalescent plasma could be the preparation of anti-SARS-CoV-2 hyperimmune globulin (hIVIG). STUDY DESIGN AND METHODS: Convalescent plasma was collected from an existing network of plasma donation centers. A caprylate/chromatography purification process was used to manufacture hIVIG. Initial batches of hIVIG were manufactured in a versatile, small-scale facility designed and built to rapidly address emerging infectious diseases. RESULTS: Processing convalescent plasma into hIVIG resulted in a highly purified immunoglobulin G (IgG) product with more concentrated neutralizing antibody activity. hIVIG will allow for the administration of greater antibody activity per unit of volume with decreased potential for several adverse events associated with plasma administration. IgG concentration and IgG specific to SARS-CoV-2 were increased over 10-fold from convalescent plasma to the final product. Normalized enzyme-linked immunosorbent assay activity (per mg/ml IgG) was maintained throughout the process. Protein content in these final product batches was 100% IgG, consisting of 98% monomer and dimer forms. Potentially hazardous proteins (IgM, IgA, and anti-A, anti-B, and anti-D) were reduced to minimal levels. CONCLUSIONS: Multiple batches of anti-SARS-CoV-2 hIVIG that met regulatory requirements were manufactured from human convalescent plasma. The first clinical study in which the hIVIG will be evaluated will be Inpatient Treatment with Anti-Coronavirus Immunoglobulin (ITAC) [NCT04546581].

Urgent Treatment for Symptomatic Carotid Stenosis: The Pittsburgh Revascularization and Treatment Emergently After Stroke (PIRATES) Protocol.

BACKGROUND: Patients with symptomatic carotid stenosis remain at high risk of early recurrent stroke without revascularization. This risk must be balanced against a higher rate of periprocedural complications associated with early revascularization. OBJECTIVE: To analyze prospectively recorded data from an institutional protocol that standardized the urgent (<48 h) treatment of patients presenting with symptomatic carotid stenosis and underwent either carotid stenting (CAS) or carotid endarterectomy (CEA). METHODS: All patients presenting over 28 mo to a comprehensive stroke center with symptomatic carotid stenosis within 48 h of index event were screened for inclusion. All patients were given dual-antiplatelet therapy. If there was clinical equipoise between CEA and CAS, patients underwent angiography and subsequently revascularization if digital subtraction angiography demonstrated ≥50% stenosis. The primary outcome was a composite of stroke or death within 30 d. RESULTS: This study included 178 patients with a diagnosis of recently symptomatic carotid stenosis; 120 patients (67%) met the criteria. A total of 59 patients underwent CEA and 61 patients underwent CAS. There were not significant differences in the primary outcome; 3 patients (5.1%) in the CEA arm and 3 patients (4.9%) in the CAS arm met the primary outcome. CONCLUSION: In this prospective analysis, urgent revascularization for symptomatic carotid stenosis can be done with equivalently low rates of stroke or death, regardless of revascularization strategy.

Immune globulin subcutaneous, human 20% solution (Xembify®), a new high concentration immunoglobulin product for subcutaneous administration.

Immune globulin subcutaneous, human 20% solution (IGSC-C 20%, Xembify®)-a new 20% immunoglobulin (IgG) liquid product for subcutaneous (SC) administration-has been developed by Grifols. The IGSC-C 20% formulation is based on knowledge acquired from the formulation of Immune Globulin Injection (Human),10% Caprylate/Chromatography Purified (IGIV-C 10%, Gamunex®-C). The protein concentration was increased from 10% to 20% to provide a smaller volume for SC administration. The IGSC-C 20% manufacturing process employs the same caprylate/chromatography purification steps as IGIV-C 10%, with the addition of an ultrafiltration step so that the product can be formulated at a higher protein concentration. IGSC-C 20% has been produced at full industrial scale to support clinical studies and licensure. These batches were characterized using a comprehensive panel of analytical testing. The new IGSC-C 20% product maintains the same composition, neutralizing activity, purity, and quality characteristics found in IGIV-C 10%.

Comparison of the liquid and lyophilized formulations of Prolastin®-C for Alpha1-Antitrypsin deficiency: Biochemical characteristics, pharmacokinetics, safety and neoantigenicity in rabbits.

Multiple analytical and preclinical studies were performed to compare the biochemical characteristics, pharmacokinetics (PK), safety and neoantigenicity of a new 5% liquid formulation of Alpha-1 Proteinase Inhibitor (Liquid A1PI, Prolastin®-C Liquid) with the lyophilized version (Lyophilized A1PI, Prolastin®-C). Liquid A1PI and Lyophilized A1PI had similar average mass (~52 kDa), and both forms exhibited glycoform patterns consistent with the known banding pattern of A1PI (dominated by the M6 and M4 bands, including deconvoluted masses). Both Liquid A1PI and Lyophilized A1PI yielded average percent purity values ranging from 96% to 99% and had active content ranging from 53  mg/mL to 59  mg/mL. The PK profile of Liquid A1PI was similar to Lyophilized A1PI. Safety assessments in rabbits showed good tolerability and no test article-related changes in mortality, clinical signs, clinical pathology, body weight, food consumption, or urinalysis parameters. Following immunodepletion of antibodies that recognize Lyophilized A1PI, there were no significant differences in the anti-drug titers among animals immunized with Lyophilized A1PI and Liquid A1PI (p > 0.05), indicating that no antibodies to neoantigens were generated. Liquid A1PI and Lyophilized A1PI have similar profiles with respect to biochemical characteristics, PK, safety and neoantigenicity.

Large-bore aspiration catheter selection does not influence reperfusion or outcome after manual aspiration thrombectomy.

INTRODUCTION: Various large-bore catheters can be employed for manual aspiration thrombectomy (MAT); clinical differences are rarely explored. METHODS: Prospectively collected demographic, angiographic, and clinical data for patients with acute internal carotid artery, middle cerebral artery M1, or basilar occlusions undergoing MAT over 23 months at a comprehensive stroke center were reviewed. We excluded patients in stentriever-based randomized trials/registries. The four most commonly utilized aspiration catheters were analyzed, and multivariate logistic regression analyses were performed to determine the effect of primary aspiration catheter choice on first-pass success, final reperfusion, and modified Rankin Scale (mRS) score at 90 days. RESULTS: Of 464 large vessel thrombectomies, 180 were performed via MAT on the first pass with one of four catheters. First-pass success was achieved in 42% of cases overall; this rate did not differ significantly between catheters: 50% for Sofia, 45% for CAT6, 40% for 0.072 inch Navien, and 36% for ACE68, p=0.67. Final Thrombolysis in Cerebral Infarction 2b or 3 reperfusion was achieved in 94% of cases overall: 97% of cases with CAT6, 95% with Sofia, 92% with Navien, and 92% with ACE68, p=0.70. Mean number of passes for index thrombus (2.0 overall), median procedure time (32 min overall), 90-day good outcome (mRS 0-2, mean 36%), and 90-day mortality (mean 27%) did not differ significantly between patients treated with different initial catheters. CONCLUSION: Among large-bore aspiration catheters, catheter selection is not an independent predictor of first-pass success, final reperfusion, or clinical outcome.

Neurologic illness in Zambia: A neurointensivist's experience.

INTRODUCTION: Management of critically ill patients in dedicated intensive care units (ICUs) is the standard of care in high income countries (HICs), but remains uncommon in low and middle-income countries (LMICs). We sought to determine the prevalence of neurologic disorders in the ICU of a LMIC and examine if resource appropriate specialized neurocritical care training could benefit these patients. METHODS: From February to March 2017, a trained neurocritical care intensivist recorded encounters in the sole ICU at the University Teaching Hospital (UTH) in Lusaka, Zambia. We stratified each patient by demographics, presence of primary or secondary neurologic deficit, comorbidities, and outcome. RESULTS: Of the 33 patients seen during this period, 26 (78.8%) had a neurologic deficit. An equal number of patients carried a primary neurologic diagnosis (13) versus a secondary neurologic diagnosis (13). Primary neurologic disorders included spinal cord injury/tumor/abscess, intracranial hemorrhage, Guillain-Barre syndrome, and traumatic brain injury. CONCLUSIONS: Over three-quarters of critically ill patients in the observation period carried a neurologic diagnosis. Future research should aim to identify if resource appropriate neurocritical care training of frontline providers may lead to improved clinical outcomes.

Spatiotemporal Mapping of Interictal Spike Propagation: A Novel Methodology Applied to Pediatric Intracranial EEG Recordings.

Synchronized cortical activity is implicated in both normative cognitive functioning and many neurologic disorders. For epilepsy patients with intractable seizures, irregular synchronization within the epileptogenic zone (EZ) is believed to provide the network substrate through which seizures initiate and propagate. Mapping the EZ prior to epilepsy surgery is critical for detecting seizure networks in order to achieve postsurgical seizure control. However, automated techniques for characterizing epileptic networks have yet to gain traction in the clinical setting. Recent advances in signal processing and spike detection have made it possible to examine the spatiotemporal propagation of interictal spike discharges across the epileptic cortex. In this study, we present a novel methodology for detecting, extracting, and visualizing spike propagation and demonstrate its potential utility as a biomarker for the EZ. Eighteen presurgical intracranial EEG recordings were obtained from pediatric patients ultimately experiencing favorable (i.e., seizure-free, n = 9) or unfavorable (i.e., seizure-persistent, n = 9) surgical outcomes. Novel algorithms were applied to extract multichannel spike discharges and visualize their spatiotemporal propagation. Quantitative analysis of spike propagation was performed using trajectory clustering and spatial autocorrelation techniques. Comparison of interictal propagation patterns revealed an increase in trajectory organization (i.e., spatial autocorrelation) among Sz-Free patients compared with Sz-Persist patients. The pathophysiological basis and clinical implications of these findings are considered.

Leaving tissue associated with infrequent intracranial EEG seizure onsets is compatible with post-operative seizure freedom.

Identify seizure onset electrodes that need to be resected for seizure freedom in children undergoing intracranial electroencephalography recording for treatment of medically refractory epilepsy. All children undergoing intracranial electroencephalography subdural grid electrode placement at the Children's Hospital of Philadelphia from 2002-2008 were asked to enroll. We utilized intraoperative pictures to determine the location of the electrodes and define the resection cavity. A total of 15 patients had surgical fields that allowed for complete identification of the electrodes over the area of resection. Eight of 15 patients were seizure free after a follow up of 1.7 to 8 yr. Only one seizure-free patient had complete resection of all seizure onset associated tissue. Seizure free patients had resection of 64.1% of the seizure onset electrode associated tissue, compared to 35.2% in the not seizure free patients (p=0.05). Resection of tissue associated with infrequent seizure onsets did not appear to be important for seizure freedom. Resecting ≥ 90% of the electrodes from the predominant seizure contacts predicted post-operative seizure freedom (p=0.007). The best predictor of seizure freedom was resecting ≥ 90% of tissue involved in majority of a patient's seizures. Resection of tissue under infrequent seizure onset electrodes was not necessary for seizure freedom.

Interictal EEG spikes identify the region of electrographic seizure onset in some, but not all, pediatric epilepsy patients.

PURPOSE: The role of sharps and spikes, interictal epileptiform discharges (IEDs), in guiding epilepsy surgery in children remains controversial, particularly with intracranial electroencephalography (IEEG). Although ictal recording is the mainstay of localizing epileptic networks for surgical resection, current practice dictates removing regions generating frequent IEDs if they are near the ictal onset zone. Indeed, past studies suggest an inconsistent relationship between IED and seizure-onset location, although these studies were based upon relatively short EEG epochs. METHODS: We employ a previously validated, computerized spike detector to measure and localize IED activity over prolonged, representative segments of IEEG recorded from 19 children with intractable, mostly extratemporal lobe epilepsy. Approximately 8 h of IEEG, randomly selected 30-min segments of continuous interictal IEEG per patient, were analyzed over all intracranial electrode contacts. RESULTS: When spike frequency was averaged over the 16-time segments, electrodes with the highest mean spike frequency were found to be within the seizure-onset region in 11 of 19 patients. There was significant variability between individual 30-min segments in these patients, indicating that large statistical samples of interictal activity were required for improved localization. Low-voltage fast EEG at seizure onset was the only clinical factor predicting IED localization to the seizure-onset region. CONCLUSIONS: Our data suggest that automated IED detection over multiple representative samples of IEEG may be of utility in planning epilepsy surgery for children with intractable epilepsy. Further research is required to better determine which patients may benefit from this technique a priori.

NMR analysis of [methyl-13C]methionine UvrB from Bacillus caldotenax reveals UvrB-domain 4 heterodimer formation in solution.

UvrB is a central DNA damage recognition protein involved in bacterial nucleotide excision repair. Structural information has been limited by the apparent disorder of the C-terminal domain 4 in crystal structures of intact UvrB; in solution, the isolated domain 4 is found to form a helix-loop-helix dimer. In order to gain insight into the behavior of UvrB in solution, we have performed NMR studies on [methyl-13C]methionine-labeled UvrB from Bacillus caldotenax (molecular mass=75 kDa). The 13 methyl resonances were assigned on the basis of site-directed mutagenesis and domain deletion. Solvent accessibility was assessed based on the relaxation and chemical shift responses of the probe methyl resonances to the stable nitroxide, 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL). M632, located at the potential dimer interface of domain 4, provides an ideal probe for UvrB dimerization behavior. The M632 resonance of UvrB is very broad, consistent with some degree of monomer-dimer exchange and/or conformational instability of the exposed dimer interface. Upon addition of unlabeled domain 4 peptide, the M632 resonance of UvrB sharpens and shifts to a position consistent with a UvrB-domain 4 heterodimer. A dissociation constant (KD) value of 3.3 microM for the binding constant of UvrB with the domain 4 peptide was derived from surface plasmon resonance studies. Due to the flexibility of the domain 3-4 linker, inferred from limited proteolysis data and from the relaxation behavior of linker residue M607, the position of domain 4 is constrained not by the stiffness of the linking segment but by direct interactions with domains 1-3 in UvrB. In summary, UvrB homodimerization is disfavored, while domain 4 homodimerization and UvrB-domain 4 heterodimerization are allowed.

Comparison of novel computer detectors and human performance for spike detection in intracranial EEG.

OBJECTIVE: Interictal spikes in intracranial EEG (iEEG) may correlate with epileptogenic cortex, but review of interictal iEEG is labor intensive. Accurate automated spike detectors are necessary for understanding the role of spikes in epileptogenesis. METHODS: The sensitivity, accuracy and reproducibility of three automated iEEG spike detectors were compared against two human EEG readers using iEEG segments from eight patients. A consensus set of detections was generated for detector calibration. Spike verification was calculated after both human EEG readers independently reviewed all detections. RESULTS: Humans and two of the three automated detectors demonstrated comparable accuracy. In four patients, automated spike detection sensitivity was >70% and accuracy was >50%. In the remaining four patients, EEG background morphology resulted in poorer performance. Blinded human verification accuracy was 76.7+/-6.6% for computer-detected spikes, and 84.5+/-4.1% for human-detected spikes. CONCLUSIONS: Automated iEEG spike detectors perform comparably to humans, but sensitivity and accuracy are patient dependent. Humans verified the majority of computer-detected spikes. SIGNIFICANCE: In some patients automated detectors may be used for mapping spike occurrences in epileptic networks. This may reveal associations between spike distribution, seizure onset, and pathology.

Structure of the 1,4-Bis(2'-deoxyadenosin-N(6)-yl)-2S,3S-butanediol intrastrand DNA cross-link arising from butadiene diepoxide in the human N-ras codon 61 sequence.

The 1,4-bis(2'-deoxyadenosin-N(6)-yl)-2S,3S-butanediol intrastrand DNA cross-link arises from the bis-alkylation of tandem N(6)-dA sites in DNA by R,R-butadiene diepoxide (BDO(2)). The oligodeoxynucleotide 5'-d(C(1)G(2)G(3)A(4)C(5)X(6)Y(7)G(8)A(9)A(10)G(11))-3'.5'-d(C(12)T(13)T(14)C(15)T(16)T(17)G(18)T(19)C(20)C(21)G(22))-3' contains the BDO(2) cross-link between the second and third adenines of the codon 61 sequence (underlined) of the human N-ras protooncogene and is named the (S,S)-BD-(61-2,3) cross-link (X,Y = cross-linked adenines). NMR analysis reveals that the cross-link is oriented in the major groove of duplex DNA. Watson-Crick base pairing is perturbed at base pair X(6).T(17), whereas base pairing is intact at base pair Y(7).T(16). The cross-link appears to exist in two conformations, in rapid exchange on the NMR time scale. In the first conformation, the beta-OH is predicted to form a hydrogen bond with T(16) O(4), whereas in the second, the beta-OH is predicted to form a hydrogen bond with T(17) O(4). In contrast to the (R,R)-BD-(61-2,3) cross-link in the same sequence (Merritt, W. K., Nechev, L. V., Scholdberg, T. A., Dean, S. M., Kiehna, S. E., Chang, J. C., Harris, T. M., Harris, C. M., Lloyd, R. S., and Stone, M. P. (2005) Biochemistry 44, 10081-10092), the anti-conformation of the two hydroxyl groups at C(beta) and C(gamma) with respect to the C(beta)-C(gamma) bond results in a decreased twist between base pairs X(6).T(17) and Y(7).T(16), and an approximate 10 degrees bending of the duplex. These conformational differences may account for the differential mutagenicity of the (S,S)- and (R,R)-BD-(61-2,3) cross-links and suggest that stereochemistry plays a role in modulating biological responses to these cross-links (Kanuri, M., Nechev, L. V., Tamura, P. J., Harris, C. M., Harris, T. M., and Lloyd, R. S. (2002) Chem. Res. Toxicol. 15, 1572-1580).

The efficacy of adjuvant platinum-based chemotherapy in Stage I uterine papillary serous carcinoma (UPSC).

OBJECTIVE: To determine the efficacy of adjuvant platinum-based chemotherapy in Stage I uterine papillary serous carcinoma (UPSC). METHODS: A retrospective multi-institutional investigation was performed to identify surgically staged patients with Stage I UPSC who were (1) treated after surgery with 3-6 courses of platinum-based chemotherapy without radiation from 1990-2003, and (2) followed for a minimum of 12 months, or until recurrence. RESULTS: Six patients (IA-2, IB-3, IC-1) were treated with carboplatin (AUC 6) or cisplatin (50 mg/m2) alone. One patient recurred to the vagina, was treated with chemo-radiation, and is alive and well at 122 months. One patient recurred to the lung, liver, and brain, and died of disease at 24 months. The remaining 4 patients are alive with no evidence of disease 15-124 months (mean 62 months) after treatment. Two patients (IB-1, IC-1) were treated with cisplatin (50 mg/m2) and cyclophosphamide (1000 mg/m2), and both are alive and well with no evidence of disease 75 and 168 months after treatment. Twenty-one patients (IA-5, IB-13, IC-3) were treated with a combination of carboplatin (AUC 6) and paclitaxel (135 mg/m2-175 mg/m2). One patient recurred to the vagina after 3 cycles of carboplatin/paclitaxel, and was treated with chemo-radiation. She is now without evidence of disease 10 months after treatment. At present, all 21 patients with Stage I UPSC treated following surgical staging with carboplatin/paclitaxel chemotherapy are alive and well with no evidence of disease 10-138 months (mean 41 months) after treatment. CONCLUSION: Combination carboplatin/paclitaxel chemotherapy following surgery is effective in the treatment of Stage I UPSC.

Structure of the 1,4-bis(2'-deoxyadenosin-N6-yl)-2R,3R-butanediol cross-link arising from alkylation of the human N-ras codon 61 by butadiene diepoxide.

The solution structure of the 1,4-bis(2'-deoxyadenosin-N(6)-yl)-2R,3R-butanediol cross-link arising from N(6)-dA alkylation of nearest-neighbor adenines by butadiene diepoxide (BDO(2)) was determined in the oligodeoxynucleotide 5'-d(CGGACXYGAAG)-3'.5'-d(CTTCTTGTCCG)-3'. This oligodeoxynucleotide contained codon 61 (underlined) of the human N-ras protooncogene. The cross-link was accommodated in the major groove of duplex DNA. At the 5'-side of the cross-link there was a break in Watson-Crick base pairing at base pair X(6).T(17), whereas at the 3'-side of the cross-link at base pair Y(7).T(16), base pairing was intact. Molecular dynamics calculations carried out using a simulated annealing protocol, and restrained by a combination of 338 interproton distance restraints obtained from (1)H NOESY data and 151 torsion angle restraints obtained from (1)H and (31)P COSY data, yielded ensembles of structures with good convergence. Helicoidal analysis indicated an increase in base pair opening at base pair X(6).T(17), accompanied by a shift in the phosphodiester backbone torsion angle beta P5'-O5'-C5'-C4' at nucleotide X(6). The rMD calculations predicted that the DNA helix was not significantly bent by the presence of the four-carbon cross-link. This was corroborated by gel mobility assays of multimers containing nonhydroxylated four-carbon N(6),N(6)-dA cross-links, which did not predict DNA bending. The rMD calculations suggested the presence of hydrogen bonding between the hydroxyl group located on the beta-carbon of the four-carbon cross-link and T(17) O(4), which perhaps stabilized the base pair opening at X(6).T(17) and protected the T(17) imino proton from solvent exchange. The opening of base pair X(6).T(17) altered base stacking patterns at the cross-link site and induced slight unwinding of the DNA duplex. The structural data are interpreted in terms of biochemical data suggesting that this cross-link is bypassed by a variety of DNA polymerases, yet is significantly mutagenic [Kanuri, M., Nechev, L. V., Tamura, P. J., Harris, C. M., Harris, T. M., and Lloyd, R. S. (2002) Chem. Res. Toxicol. 15, 1572-1580].

Dual roles of glycosyl torsion angle conformation and stereochemical configuration in butadiene oxide-derived N1 beta-hydroxyalkyl deoxyinosine adducts: a structural perspective.

The solution structure of the N1-[1-hydroxy-3-buten-2(R)-yl]-2'-deoxyinosine adduct arising from the alkylation of adenine N1 by butadiene epoxide (BDO), followed by deamination to deoxyinosine, was determined in the oligodeoxynucleotide 5'-d(CGGACXAGAAG)-3'.5'-d(CTTCTTGTCCG)-3'. This oligodeoxynucleotide contained the BDO adduct at the second position of codon 61 of the human N-ras protooncogene (underlined) and was named the ras61 R-N1-BDO-(61,2) adduct. 1H NMR revealed a weak C5 H1' to X6 H8 nuclear Overhauser effects (NOE), followed by an intense X6 H8 to X6 H1' NOE. Simultaneously, the X6 H8 to X6 H3' NOE was weak. The resonances arising from the T16 and T17 imino protons were not observed. 1H NOEs between the butadiene moiety and the DNA positioned the adduct in the major groove. Structural refinement based upon a total of 394 NOE-derived distance restraints and 151 torsion angle restraints yielded a structure in which the modified deoxyinosine was in the syn conformation about the glycosyl bond, with a glycosyl bond angle of 83 degrees , and T17, the complementary nucleotide, was stacked into the helix but not hydrogen bonded with the adducted inosine. The refined structure provides a plausible hypothesis as to why these N1 deoxyinosine adducts strongly code for the incorporation of dCTP during trans lesion DNA replication, irrespective of stereochemistry, both in Escherichia coli [Rodriguez, D. A., Kowalczyk, A., Ward, J. B. J., Harris, C. M., Harris, T. M., and Lloyd, R. S. (2001) Environ. Mol. Mutagen. 38, 292-296] and in mammalian cells [Kanuri, M., Nechev, L. N., Tamura, P. J., Harris, C. M., Harris, T. M., and Lloyd, R. S. (2002) Chem. Res. Toxicol. 15, 1572-1580]. Rotation of the N1 deoxyinosine adduct into the syn conformation may facilitate incorporation of dCTP via Hoogsteen type templating with deoxyinosine, generating A to G mutations. However, conformational differences between the R- and the S-N1-BDO-(61,2) adducts, involving the positioning of the butenyl moiety in the major groove of DNA, suggest that adduct stereochemistry plays a secondary role in modulating the biological response to these adducts.

Structure of an oligodeoxynucleotide containing a butadiene oxide-derived N1 beta-hydroxyalkyl deoxyinosine adduct in the human N-ras codon 61 sequence.

The solution structure of the N1-(1-hydroxy-3-buten-2(S)-yl)-2'-deoxyinosine adduct arising from the alkylation of adenine N1 by butadiene epoxide (BDO), followed by deamination to deoxyinosine, was determined, in the oligodeoxynucleotide d(CGGACXAGAAG).d(CTTCTCGTCCG). This oligodeoxynucleotide contained the BDO adduct at the second position of codon 61 of the human N-ras protooncogene, and was named the ras61 S-N1-BDO-(61,2) adduct. (1)H NMR revealed a weak C(5) H1' to X(6) H8 NOE, followed by an intense X(6) H8 to X(6) H1' NOE. Simultaneously, the X(6) H8 to X(6) H3' NOE was weak. The resonance arising from the T(17) imino proton was not observed. (1)H NOEs between the butadiene moiety and the DNA positioned the adduct in the major groove. Structural refinement based upon a total of 364 NOE-derived distance restraints yielded a structure in which the modified deoxyinosine was in the high syn conformation about the glycosyl bond, and T(17), the complementary nucleotide, was stacked into the helix, but not hydrogen bonded with the adducted inosine. The refined structure provided a plausible hypothesis as to why this N1 deoxyinosine adduct strongly coded for the incorporation of dCTP during trans lesion DNA replication, both in Escherichia coli [Rodriguez, D. A., Kowalczyk, A., Ward, J. B. J., Harris, C. M., Harris, T. M., and Lloyd, R. S. (2001) Environ. Mol. Mutagen. 38, 292-296], and in mammalian cells [Kanuri, M., Nechev, L. N., Tamura, P. J., Harris, C. M., Harris, T. M., and Lloyd, R. S. (2002) Chem. Res. Toxicol. 15, 1572-1580]. Rotation of the N1 deoxyinosine adduct into the high syn conformation may facilitate incorporation of dCTP via Hoogsteen-type templating with deoxyinosine, thus generating A-to-G mutations.