Excessive intragastric alcohol administration exacerbates hepatic encephalopathy and provokes neuronal cell death in male rats with chronic liver disease.

Hepatic encephalopathy (HE) is defined as decline in neurological function during chronic liver disease (CLD). Alcohol is a major etiological factor in the pathogenesis of fibrosis/cirrhosis and has also been documented to directly impact the brain. However, the role of alcohol in the development of HE in CLD remains unclear. Here, we investigated the impact of excessive alcohol administration on neurological deterioration in rats with CLD. Starting day 7 post-BDL surgery, rats were administered alcohol twice daily (51% v/v ethanol, 3 g/kg, via gavage) for 4 weeks. Motor coordination was assessed weekly using rotarod and anxiety-like behavior was evaluated with open field and elevated plus maze at 5 weeks. Upon sacrifice, brains were collected for western blot and immunohistochemical analyses to investigate neuronal integrity and oxidative stress status. Alcohol worsened motor coordination performance and increased anxiety-like behavior in BDL rats. Impairments were associated with decreased neuronal markers of NeuN and SMI311, increased apoptotic markers of cleaved/pro-caspase-3 and Bax/Bcl2, increased necroptosis markers of pRIP3 and pMLKL, decreased total antioxidant capacity (TAC), and increased 4-hydroxynonenal (4-HNE)modified proteins in the cerebellum of BDL-alcohol rats when compared to respective controls. Immunofluorescence confirmed the colocalization of cleaved caspase-3 and pMLKL in the granular neurons of the cerebellum of BDL-alcohol rats. Excessive alcohol consumption exacerbates HE which leads to associated apoptotic and necroptotic neuronal loss in the cerebellum of BDL-alcohol rats. Additionally, higher levels of 4-HNE and decreased TAC in the cerebellum of BDL-alcohol rats suggest oxidative stress is the triggering factor of apoptotic and necroptotic neuronal loss/injury.

Development of an Evaluation System for the Prophylactic Use of Antimicrobial Drugs in the Perioperative Period of Class I Surgical Incisions in Neurosurgery.

BACKGROUND: This study aimed to establish a precise preoperative high-risk factor scoring system and algorithm for antibiotic prophylaxis decision-making, provide guidance for the judicious use of AMP, refine interventions, and ensure the appropriate application of AMP for class I incisions in neurosurgery. METHODS: According to PRISMA guidelines, literature searches, study selection, methodology development, and quality appraisal were performed. The quality of evidence across the study population was assessed using the Newcastle-Ottawa Scale. A two-round Delphi expert consultation method involved 15 experts from leading tertiary hospitals in China. Establishing an algorithm of SOPs for perioperative antimicrobial prophylaxis in Class I neurosurgical incisions. RESULTS: Thirteen studies, encompassing 11,936 patients undergoing clean neurosurgical procedures, were included. 791 patients experienced SSI, resulting in an average incidence of 6.62%. Identified risk factors significantly associated with an increased incidence of postoperative SSI (P < 0.05) included emergency surgery, preoperative hospitalization ≥7 days, intraoperative blood loss ≥300 mL, operation time ≥4 hours, diabetes mellitus, cerebrospinal fluid leakage, and repeat surgery. Sensitivity analysis demonstrated robust results for emergency surgery, intraoperative blood loss ≥300 mL, operation time ≥4 hours, cerebrospinal fluid leakage, and repeat surgery. Established a risk assessment system for Class I neurosurgical incisions by the Delphi method. Additionally, we have formulated an algorithm of SOPs for perioperative antimicrobial prophylaxis in Class I neurosurgical incisions. CONCLUSIONS: The established index for AMP utilization and SOPs in the preoperative period of class I neurosurgical incisions proves valuable, contributing to improved patient outcomes in neurosurgical procedures.

Regulation of eukaryotic protein kinases by Pin1, a peptidyl-prolyl isomerase.

The peptidyl-prolyl isomerase Pin1 cooperates with proline-directed kinases and phosphatases to regulate multiple oncogenic pathways. Pin1 specifically recognizes phosphorylated Ser/Thr-Pro motifs in proteins and catalyzes their cis-trans isomerization. The Pin1-catalyzed conformational changes determine the stability, activity, and subcellular localization of numerous protein substrates. We conducted a survey of eukaryotic protein kinases that are regulated by Pin1 and whose Pin1 binding sites have been identified. Our analyses reveal that Pin1 target sites in kinases do not fall exclusively within the intrinsically disordered regions of these enzymes. Rather, they fall into three groups based on their location: (i) within the catalytic kinase domain, (ii) in the C-terminal kinase region, and (iii) in regulatory domains. Some of the kinases downregulated by Pin1 activity are tumor-suppressing, and all kinases upregulated by Pin1 activity are functionally pro-oncogenic. These findings further reinforce the rationale for developing Pin1-specific inhibitors as attractive pharmaceuticals for cancer therapy.

Effects of Enzymatic Cottonseed Protein Concentrate as a Feed Protein Source on the Growth, Plasma Parameters, Liver Antioxidant Capacity and Immune Status of Largemouth Bass (Micropterus salmoides).

This study appraised the impact of enzymatic cottonseed protein concentrate (ECP) as a fish meal (FM) substitute on the growth and health of largemouth bass (Micropterus salmoides) (initial weight 14.99 ± 0.03 g). Five diets with equal nitrogen, fat, and energy were designed to replace 0%, 7.78%, 15.56%, 23.33%, and 31.11% FM by adding 0%, 3.6%, 7.2%, 10.8%, and 14.4% ECP, named ECP0, ECP3.6, ECP7.2, ECP10.8, and ECP14.4, respectively. We fed 300 fish with five experimental diets for 60 days. The results revealed that weight gain rate (WGR) and specific growth rate (SGR) did not notably reduce until the addition of ECP exceeded 7.2%. The proximate composition of fish was not affected by the amount of ECP added in diets. Plasma total protein (TP), albumin (ALB), and high-density lipoprotein (HDL) concentrations increased with the increase of ECP dosage, while the triglyceride (TG) and low-density lipoprotein (LDL) concentrations and alkaline phosphatase (ALP) activity showed an opposite trend. For hepatic antioxidant capacity, the hepatic total superoxide dismutase (T-SOD) and catalase (CAT) activities, glutathione (GSH) content, and the expression of nuclear factor erythroid 2-related factor 2 (Nrf2), superoxide dismutase (SOD), and CAT were increased by ECP, while the hepatic malondialdehyde (MDA) content and the expression of kelch-like-ECH-associated protein 1 (Keap1) were decreased. With regard to inflammation, the expression of nuclear factor-kappa B (NF-κB), interleukin-8 (IL-8), and tumor necrosis factor-α (TNF-α) were inhibited by ECP. In summary, the amount of ECP added to diet can reach 7.2% to replace 15.56% FM without hampering the growth of largemouth bass, and ECP can improve the antioxidant and immune capacity.

Effects of phytase supplementation of high-plant-protein diets on growth, phosphorus utilization, antioxidant, and digestion in red swamp crayfish (Procambarus clarkii).

Fish meal is increasingly being replaced by plant protein raw materials, meanwhile, it brings phytic acid, which combines with phosphorus to form phytate phosphorus and leads to a low utilization rate of phosphorus in shrimp. To solve this problem, this study investigated the effects of phytase supplementation on growth performance, phosphorus utilization, antioxidants, and digestion in red swamp crayfish (Procambarus clarkii). Crayfish (initial mean weight: 8.69 ± 0.15 g, N = 324) were randomly divided into six groups each with three replicates of 18 individuals each, and hand-fed for 8 weeks with one of six experimental diets (50 and 490 g kg-1 animal and plant protein raw material, respectively): negative control (NC; 11.0 g kg-1 phosphorus), positive control (PC; 15 g kg-1 NaH2PO4 added to NC; 14.7 g kg-1 phosphorus), and phytase supplementation diets (P1-P4: 0.1, 0.2, 0.4, and 0.6 g kg-1 phytase added to NC, respectively). The feeding trial was performed in a micro-flow water culture system. P2 showed a significantly higher weight gain rate (WGR), specific growth rate, protein efficiency ratio, and protein retention efficiency (PRE) but showed the lowest feed conversion ratio (FCR) than other groups. Broken-line regression analyses using WGR, FCR, and PRE as evaluation indices showed that the optimal dietary phytase supplementation level was 0.233, 0.244, and 0.303 g kg-1, respectively. P2 showed the highest crude protein content of whole crayfish and abdominal muscle, and phosphorus deposition rate, which was significantly higher than that in NC and PC. P3 showed the highest calcium and phosphorus contents in whole crayfish and phosphorus content in abdominal muscle, and calcium and inorganic phosphorus content in serum, which were significantly higher than those in NC. P3 showed significantly lowest serum alkaline phosphatase, alanine aminotransferase, aspartate transaminase activities, malondialdehyde content in hepatopancreas, and highest catalase activity, which were significantly lower and higher, respectively, than those in NC and PC. In summary, the addition of 0.2-0.4 g kg-1 phytase significantly improves the growth performance, feed utilization, digestive enzyme activity, and antioxidant of P. clarkii, which has a similar effect to the direct addition of NaH2PO4 at 15 g kg-1 to the feed.

Dietary chlorella (Chlorella vulgaris) supplementation effectively improves body color, alleviates muscle inflammation and inhibits apoptosis in largemouth bass (Micropterus salmoides).

Muscle quality, antioxidant status, and inflammatory and apoptotic molecule expression were investigated in juvenile largemouth bass fed five levels of Chlorella for 60 days. The results showed that muscle quality can be improved by increasing the muscle crude protein content, muscle and skin brightness value (L*), redness value (a*) and yellowness value (b*) in Chlorella-supplemented diets without affecting the growth and muscle fiber development of fish. Chlorella supplementation did not cause oxidative stress in muscle, but optimal Chlorella administration alleviated the muscle inflammatory response by downregulating the nuclear factor κB (NF-κB)-mediated proinflammatory factors such as interleukin 1ß (IL-1ß) and interleukin 8 (IL-8). Moreover, anti-apoptotic effects were induced by upregulation of anti-apoptotic genes, such as b cell lymphoma-2 (bcl-2) and myeloid cell leukemia-1 (mcl-1), and downregulation of pro-apoptotic genes, including bcl2-associated x (bax) and caspase3. In conclusion, Chlorella improved muscle quality, alleviated muscle inflammation and resisted muscle apoptosis.

POGZ promotes homology-directed DNA repair in an HP1-dependent manner.

The heterochromatin protein HP1 plays a central role in the maintenance of genome stability but little is known about how HP1 is controlled. Here, we show that the zinc finger protein POGZ promotes the presence of HP1 at DNA double-strand breaks (DSBs) in human cells. POGZ depletion delays the resolution of DSBs and sensitizes cells to different DNA-damaging agents, including cisplatin and talazoparib. Mechanistically, POGZ promotes homology-directed DNA repair by retaining the BRCA1/BARD1 complex at DSBs in an HP1-dependent manner. In vivo CRISPR inactivation of Pogz is embryonically lethal. Pogz haploinsufficiency (Pogz+ /delta) results in developmental delay, impaired intellectual abilities, hyperactive behaviour and a compromised humoral immune response in mice, recapitulating the main clinical features of the White Sutton syndrome (WHSUS). Pogz+ /delta mice are further radiosensitive and accumulate DSBs in diverse tissues, including the spleen and brain. Altogether, our findings identify POGZ as an important player in homology-directed DNA repair both in vitro and in vivo.

Culture salinity modulates Nrf2 antioxidant signaling pathway and immune response of juvenile Genetically Improved Farmed Tilapia (GIFT) (Oreochromis niloticus) under different dietary protein levels.

This study aimed to evaluate that dietary protein levels and culture salinity levels affect the health status of juvenile genetically improved farmed tilapia (GIFT, Oreochromis niloticus). Graded protein levels of six diets were prepared, ranging from 18.20% to 49.49% (dry basis), and were used in cultured GIFT at two salinity levels (0‰ and 8‰) for 8 weeks. The results suggested that appropriate protein levels reduced pro-inflammatory gene expressions in the intestine including interleukin 1ß (IL-1ß), interleukin 8 (IL-8) and tumour necrosis factor-α (TNF-α) mRNA levels at two salinity levels (P < 0.05). 8‰ salinity significantly decreased the expression levels of IL-1ß, TNF-α and nuclear factor-kappa B (NF-κB) (P < 0.05). The anti-inflammatory factor interleukin 10 (IL-10) was significantly increased by 36.42% protein level (P < 0.05). Regarding antioxidant capacity, appropriate protein levels and 8‰ salinity significantly improved the antioxidant capacity of fish by regulating the activities of intestinal total superoxide dismutase (T-SOD), glutathione peroxidase (GPx), and the levels of glutathione (GSH) and malondialdehyde (MDA). Furthermore, appropriate protein levels and 8‰ salinity also significantly enhanced the antioxidant gene expressions associated with the Nrf2/keap1 signaling pathway by regulating the expression levels of heme oxygenase-1 (HO-1), GPx, catalase (CAT) and superoxide dismutase (SOD). According to GPx activities and the mRNA levels of IL-10, the optimum dietary protein levels for GIFT juveniles were 31.12%-32.18% (0‰) and 34.25-35.38% (8‰) based on second-degree polynomial regression analysis. The present study found that appropriate protein levels and 8‰ culture salinity are critical in maintaining the health of GIFT juveniles by improving antioxidant and immune capacity.

Non-viral delivery of an optogenetic tool into cells with self-healing hydrogel.

Optogenetics offers unique, temporally precise control of neural activity in genetically targeted specific neurons that express light-sensitive opsin molecules. Three-dimensional (3D) delivery of optogenetics can be realized by co-injection of bacteriorhodopsin (HEBR) plasmid with a chitosan-based self-healing hydrogel with strong shear-thinning properties. The HEBR protein shows photoelectrical properties and can be used as an optical switch for cell activation. We optimize the shear force generated during the process of injection (∼100 Pa), which is transient because of the self-healing nature of the hydrogel. This transient force exerted by the self-healing hydrogel may allow the cytosolic delivery of HEBR plasmid with excellent cell viability and a high efficiency approaching 80%. When excited with green light, HEBR-delivered neural stem cells (NSCs) can proliferate and specifically differentiate into neurons in vitro and rescue the function of nerve impaired zebrafish in vivo. This novel optogenetic method combining 3D injectable self-healing hydrogel offers potential temporal-spatial approaches to treat neurodegenerative diseases in the future.

Characterization of the anti-CD22 targeted therapy, moxetumomab pasudotox, for B-cell precursor acute lymphoblastic leukemia.

Moxetumomab pasudotox is a second-generation recombinant immunotoxin against CD22 on B-cell lineages. Antileukemic activity has been demonstrated in children with chemotherapy-refractory B-cell precursor acute lymphoblastic leukemia (BCP-ALL), with variable responses. Here, we report in vitro and in vivo evaluation of moxetumomab pasudotox treatment of human cell lines and patient-derived cells as a preliminary study to understand characteristics of sensitivity to treatment. Binding, internalization, and apoptosis were evaluated using fluorescently tagged moxetumomab pasudotox. Studies in NOD-scid IL2Rgnull mice showed a modest survival benefit in mice engrafted with 697 cells but not in NALM6 or the two patient-derived xenograft models.

Circulating tumor cells: application as a biomarker for molecular characterization and predictor of survival in an all-comer solid tumor phase I clinical study.

PURPOSE: Clinical development of cancer drugs has a low success rate. Prognostic and predictive biomarkers using minimally invasive approaches hold promise for increasing the probability of success by enabling disease characterization, patient selection and early detection of drug treatment effect. Enumeration and molecular characterization of circulating tumor cells (CTC) may address some of these needs, and thus were evaluated for utility in a Phase I solid tumor clinical study. EXPERIMENTAL DESIGN: Blood samples for CTC analysis were obtained from 24 cancer patients in a multi-center all-comer Phase I study of MEDI-575, a novel anti-PDGFRα antibody. Samples were taken at screening and analyzed for enumeration of CTC using the CellSearch(®) platform and for molecular characterization using a novel quantitative RT-PCR assay. RESULTS: Fifty-nine percent of the patients showed at least 1 CTC per 7.5 ml of blood at baseline. Progression-free survival (PFS) and overall survival (OS) of patients with 0 CTCs at baseline were longer than PFS and Os for patients with 1-3 and >3 CTCs (8.8 versus 1.4 and 1.3 months PFS, P = 0.02; 9.0 vs 7.4 and 3.5 months OS, P = 0.20, respectively). Patients with 0 CTC showed a greater percentage of stable disease than the other 2 groups with 1-3 and >3 CTCs (57% vs 29% and 0%). The multimarker qRT-PCR method detected CTC in 40% of the patients, and 80% of these patients were positive for pre-selected drug target genes. CONCLUSION: CTC enumeration of patients in an all-comer study is feasible and may allow for patient stratification for PFS and Os to evaluate the clinical response of investigational agents. Gene expression profiling of isolated CTC may provide a means for molecular characterization of selected tumor targets.

Specific inhibition of the JNK pathway promotes locomotor recovery and neuroprotection after mouse spinal cord injury.

Limiting the development of secondary damage represents one of the major goals of neuroprotective therapies after spinal cord injury. Here, we demonstrate that specific JNK inhibition via a single intraperitoneal injection of the cell permeable peptide D-JNKI1 6h after lesion improves locomotor recovery assessed by both the footprint and the BMS tests up to 4 months post-injury in mice. JNK inhibition prevents c-jun phosphorylation and caspase-3 cleavage, has neuroprotective effects and results in an increased sparing of white matter at the lesion site. Lastly, D-JNKI1 treated animals show a lower increase of erythrocyte extravasation and blood brain barrier permeability, thus indicating protection of the vascular system. In total, these results clearly point out JNK inhibition as a promising neuroprotective strategy for preventing the evolution of secondary damage after spinal cord injury.

Inhibition of transmethylation down-regulates CD4 T cell activation and curtails development of autoimmunity in a model system.

Transmethylation affects several cellular events, including T cell activation, and blockade of this pathway may curtail inflammatory/autoimmune responses. Here, we demonstrate that transmethylation inhibition by a novel reversible S-adenosyl-l-homocysteine hydrolase inhibitor leads to immunosuppression by reducing phosphorylation of several key proteins involved in TCR signaling, including Akt, Erk1/2, and NF-kappaB. Remarkably, this effect was largely restricted to CD4 T cells and correlated with reduced arginine methylation of Vav1, an essential guanine nucleotide exchange factor in T cell stimulation. Treatment with the transmethylation inhibitor averted, and even ameliorated, the CD4-mediated autoimmune disease, experimental autoimmune encephalomyelitis. The data suggest that transmethylation is required for CD4 T cell activation, and its inhibition may be a novel approach in the treatment of multiple sclerosis, and other CD4-mediated autoimmune diseases.

Mechanism of nicotine-induced pulmonary fibroblast transdifferentiation.

We tested the hypothesis that in vitro nicotine exposure disrupts specific epithelial-mesenchymal paracrine signaling pathways and results in pulmonary interstitial lipofibroblast (LIF)-to-myofibroblast (MYF) transdifferentiation, resulting in altered pulmonary development and function. Studies were done to determine whether nicotine induces LIF-to-MYF transdifferentiation and to elucidate underlying molecular mechanism(s) involved and to determine whether nicotine-induced LIF-to-MYF transdifferentiation could be prevented by stimulating specific alveolar interstitial fibroblast lipogenic pathway. WI38 cells, a human embryonic pulmonary fibroblast cell line, were treated with nicotine with or without specific agonists of alveolar fibroblast lipogenic pathway, PTHrP, DBcAMP, or the potent PPARgamma stimulant rosiglitazone (RGZ) for 7 days. Expression of key lipogenic and myogenic markers was examined by RT-PCR, Western hybridization, and immunohistochemistry. The effect of nicotine on triglyceride uptake by WI38 cells and PTHrP binding to its receptor was also determined. Finally, the effect of transfecting WI38 cells with a PPARgamma expression vector on nicotine-induced LIF-to-MYF transdifferentiation was determined. Nicotine treatment resulted in significantly decreased expression of lipogenic and increased expression of myogenic markers in a dose-dependent manner, indicating nicotine-induced LIF-to-MYF transdifferentiation. This was accompanied by decreased PTHrP receptor binding to its receptor. The nicotine-induced LIF-to-MYF transdifferentiation was completely prevented by concomitant treatment with PTHrP, DBcAMP, RGZ, and by transiently overexpressing PPARgamma. Our data suggest nicotine induces alveolar LIF-to-MYF transdifferentiation through a mechanism involving downregulation of lipogenic PTHrP-mediated, cAMP-dependent PKA signaling pathway, which can be prevented using specific molecular targets. Potential therapeutic implications of these observations against in utero nicotine-induced lung injury remain to be tested.

Inhibition of S-adenosyl-L-homocysteine hydrolase induces immunosuppression.

Lymphocytes depend on transmethylation reactions for efficient activation and function. These reactions are primarily catalyzed by S-adenosylmethionine-dependent methyltransferases, which convert S-adenosylmethionine to S-adenosyl-L-homocysteine. S-adenosyl-L-homocysteine is then hydrolyzed by S-adenosyl-L-homocysteine hydrolase to prevent feedback inhibition of transmethylation reactions. By impeding S-adenosyl-L-homocysteine hydrolase, a build-up of S-adenosyl-L-homocysteine occurs, and most intracellular transmethylation reactions cease. Thus, a nontoxic inhibitor of this enzyme might be a useful immunosuppressive therapeutic agent. We identified a potent reversible type III inhibitor of S-adenosyl-L-homocysteine hydrolase, DZ2002 [methyl 4-(adenin-9-yl)-2-hydroxybutanoate], and determined its cytotoxic and immunologic effects. We demonstrated that DZ2002 blocked S-adenosyl-L-homocysteine hydrolase more effectively than a type I inhibitor, but cytotoxicity from DZ2002 was greatly reduced. Although DZ2002 did not prevent concanavalin A-induced T cell proliferation or interleukin (IL)-2 production, it significantly reduced both a mixed lymphocyte reaction and IL-12 production from in vitro-stimulated splenocytes. In addition, levels of CD80 and CD86 on human monocytic THP-1 cells were decreased in a dose-dependent manner in the presence of 0.1 to 10 microM DZ2002, and decreases were also seen in IL-12 and tumor necrosis factor-alpha production from both mouse thioglycollate-stimulated peritoneal macrophages and THP-1 cells. In vivo, DZ2002 significantly suppressed a delayed-type hypersensitivity reaction as well as antibody secretion. We conclude that DZ2002's immunosuppressive effects are likely not solely attributed to T cell inhibition but also to the obstruction of macrophage activation and function through reductions in cytokine output and/or T cell costimulation. These data suggest an important dual role for the S-adenosyl-l-homocysteine hydrolase in both macrophage and T cell function.

Closing of the nucleotide pocket of kinesin-family motors upon binding to microtubules.

We have used adenosine diphosphate analogs containing electron paramagnetic resonance (EPR) spin moieties and EPR spectroscopy to show that the nucleotide-binding site of kinesin-family motors closes when the motor.diphosphate complex binds to microtubules. Structural analyses demonstrate that a domain movement in the switch 1 region at the nucleotide site, homologous to domain movements in the switch 1 region in the G proteins [heterotrimeric guanine nucleotide-binding proteins], explains the EPR data. The switch movement primes the motor both for the free energy-yielding nucleotide hydrolysis reaction and for subsequent conformational changes that are crucial for the generation of force and directed motion along the microtubule.

A novel restricted photoaffinity spin-labeled non-nucleoside ATP analogue as a covalently attached reporter group of the active site of Myosin subfragment 1.

The photoaffinity spin-labeled non-nucleoside ATP analogue, 2-(4-azido-2-nitrophenyl)amino-2,2-(1-oxyl-2,2,6,6-tetramethyl-4-piperidylidene)di(oxymethylene)ethyl triphosphate (SSL-NANTP), has been shown to be a substrate for skeletal mysoin subfragment 1 (S1) that can be photoincorporated at the active site of S1 [Chen, X., et al. (2000) Bioconjugate Chem. 11, 725-733]. Electron paramagnetic resonance spectroscopy shows that the probe undergoes restricted motion with respect to the protein. The parent compound, NANTP (2-[(4-azido-2-nitrophenyl)amino]ethyl triphosphate), is specifically photoincorporated at Trp-130 on the amino-terminal 23 kDa tryptic fragment in rabbit skeletal myosin. Surprisingly, amino acid sequence analysis shows that SSL-NANTP is photoincorporated on the carboxy-terminal 20 kDa tryptic fragment at Lys-681 on the side opposite Trp-130 in the nucleotide pocket. This is the first direct evidence showing that this residue in the 20 kDa tryptic fragment is close enough to the active site to be photolabeled by trapped ATP analogues. After actin treatment in the presence of MgATP, SSL-NANDP-labeled myosin S1 had normal ATPase activity, indicating that photolabeling did not significantly alter the enzymatic properties of S1. Photoincorporated SSL-NANDP was bound inside the nucleotide site of S1, with an effective concentration of 20 mM as judged by the concentration of MgADP needed to displace it. Molecular dynamics simulations suggest that the ability of NANTP and SSL-NANTP to photolabel different sites results from different orientations of the phenyl ring in the active site. For SSL-NANTP, the p-azido group on the phenyl ring points toward Lys-681. For NANTP, it points in the opposite direction toward Trp-130.