[Clinical and imaging features of acute encephalopathy with biphasic seizures and late reduced diffusion in children].

Objective: To explore the clinical and imaging features of acute encephalopathy with biphasic seizures and late reduced diffusion(AESD) in children. Methods: For the case series study, 21 children with AESD from Peking University First Hospital, Provincial Children's Hospital Affiliated to Anhui Medical University, Children's Hospital of Fudan University, and Shanxi Children's Hospital who were diagnosed and treated from October 2021 to July 2023 were selected. Clinical data were collected to summarize their clinical information, imaging, and laboratory tests, as well as treatment and prognostic characteristics. Descriptive statistical analysis was applicated. Results: Of the 21 cases with AESD, 11 were males and 10 were females, with the age of onset of 2 years and 6 months (1 year and 7 months, 3 years and 6 months). Of the 21 cases, 18 were typical cases with biphasic seizures. All typical cases had early seizures within 24 hours before or after fever onset. Among them, 16 cases had generalized seizures, 2 cases had focal seizures, and 7 cases reached the status epilepticus. Of the 21 cases, 3 atypical cases had late seizures in biphasic only. The late seizures in the 21 cases occurred on days 3 to 9. The types of late seizures included focal seizures in 12 cases, generalized seizures in 6 cases, and both focal and generalized seizures in 3 cases. Diffusion-weighted imaging (DWI) test on days 3 to 11 showed reduced diffusion of subcortical white matter which was named "bright tree sign" in all cases. The diffuse cerebral atrophy predominantly presented in the front-parietal-temporal lobes was found in 19 cases between day 12 and 3 months after the onset of the disease. Among 21 cases, 20 had been misdiagnosed as autoimmune encephalitis, central nervous system infection, febrile convulsions, posterior reversible encephalopathy syndrome, acute disseminated encephalomyelitis, and hemiconvulsion-hemiplegia-epilepsy syndrome. All the cases received high-dose gammaglobulin and methylprednisolone pulse therapy with poor therapeutic effect. By July 2023, 18 cases were under follow-up. Among them, 17 cases were left with varying degrees of neurologic sequelae, including 11 cases with post-encephalopathic epilepsy; 1 recovered completely. Conclusions: AESD is characterized by biphasic seizures clinically and "bright tree sign" on DWI images. Symptomatic and supportive treatments are recommended. The immunotherapy is ineffective. The prognosis of AESD is poor, with a high incidence of neurological sequelae and a low mortality.

[Clinical and genetic characteristics of 9 rare cases with coexistence of dual genetic diagnoses].

Objective: To analyze the clinical and genetic characteristics of pediatric patients with dual genetic diagnoses (DGD). Methods: Clinical and genetic data of pediatric patients with DGD from January 2021 to February 2022 in Peking University First Hospital were collected and analyzed retrospectively. Results: Among the 9 children, 6 were boys and 3 were girls. The age of last visit or follow-up was 5.0 (2.7,6.8) years. The main clinical manifestations included motor retardation, mental retardation, multiple malformations, and skeletal deformity. Cases 1-4 were all all boys, showed myopathic gait, poor running and jumping, and significantly increased level of serum creatine kinase. Disease-causing variations in Duchenne muscular dystrophy (DMD) gene were confirmed by genetic testing. The 4 children were diagnosed with DMD or Becker muscular dystrophy combined with a second genetic disease, including hypertrophic osteoarthropathy, spinal muscular atrophy, fragile X syndrome, and cerebral cavernous malformations type 3, respectively. Cases 5-9 were clinically and genetically diagnosed as COL9A1 gene-related multiple epiphyseal dysplasia type 6 combined with NF1 gene-related neurofibromatosis type 1, COL6A3 gene-related Bethlem myopathy with WNT1 gene-related osteogenesis imperfecta type XV, Turner syndrome (45, X0/46, XX chimera) with TH gene-related Segawa syndrome, Chromosome 22q11.2 microduplication syndrome with DYNC1H1 gene-related autosomal dominant lower extremity-predominant spinal muscular atrophy-1, and ANKRD11 gene-related KBG syndrome combined with IRF2BPL gene-related neurodevelopmental disorder with regression, abnormal movement, language loss and epilepsy. DMD was the most common, and there were 6 autosomal dominant diseases caused by de novo heterozygous pathogenic variations. Conclusions: Pediatric patients with coexistence of double genetic diagnoses show complex phenotypes. When the clinical manifestations and progression are not fully consistent with the diagnosed rare genetic disease, a second rare genetic disease should be considered, and autosomal dominant diseases caused by de novo heterozygous pathogenic variation should be paid attention to. Trio-based whole-exome sequencing combining a variety of molecular genetic tests would be helpful for precise diagnosis.

[Analysis of 4 children with DYNC1H1 gene related spinal muscular atrophy with lower extremity predominant 1].

Objective: To investigate the clinical features and gene variation characteristics of children with dynein cytoplasmic 1 heavy chain 1 (DYNC1H1) gene associated spinal muscular atrophy with lower extremity predominant (SMALED) 1. Methods: The clinical data of 4 SMALED1 children admitted to Peking University First Hospital from December 2018 to May 2021, who were found to have pathogenic variation of DYNC1H1 gene through genetic testing, except for other genes known to be related to motor retardation, were retrospectively summarized to analyze the phenotype and genotype characteristics. Results: There were 3 males and 1 female. The age of onset was 1 year, 1 day, 1 day and 4 months, respectively. The age of diagnosis was 4 years and 10 months, 9 months, 5 years and 9 months, and 3 years and 1 month, respectively. The clinical manifestations were muscle weakness and muscular atrophy of lower limbs, 2 cases with foot deformity, 1 case with early non progressive joint contracture, 1 case with hip dislocation and 1 case with mental retardation. De novo heterozygous missense variations in DYNC1H1 gene were found in all 4 children. According to the rating of American College of medical genetics and genomics, they were all possible pathogenic and pathogenic variations, with p.R598C, p.P776L, p.Y1109D variations had been reported, and p.I1086R variation had not been reported. Conclusions: For those with unexplained lower limb muscle weakness, muscle atrophy, joint contracture and foot deformity, upper limb motor ability related retention, with or without mental retardation, as well as the motor ability progresses slowly, it is necessary to consider the possibility of SMALED1 and the detection of DYNC1H1 gene when necessary.

[A family of hereditary protein S deficiency with the onset of pulmonary embolism and literature review].

Objective: To explore the clinical characteristics and genotype of PROS1 gene related hereditary protein S deficiency (PSD) with the onset of pulmonary embolism in children. Methods: A family with pulmonary embolism was diagnosed as hereditary PSD in the Department of Pediatrics of Peking University First Hospital in November 2020, and the clinical data, including clinical manifestations, laboratory tests, imaging and genetic results, were collected for a retrospective research. The family members were also screened for protein S activity and PROS1 gene mutations. A literature search with "PROS1" "protein S deficiency" "homozygous" and "complex heterozygous" as key words was conducted at PubMed, China National Knowledge Infrastructure, and Wanfang Data Knowledge Service Platform (up to October 2021). Case reports of patients with PROS1 gene homozygous or complex heterozygous variants and related clinical features, protein S activity, and genotype were reviewed and analyzed. Results: The proband, a 14-year-old girl, was admitted to the hospital for a 9-day history of coughing and a 4-day history of chest pain in November 2020. After admission, laboratory tests showed that D-dimer was 8.38 mg/L (reference:<0.24 mg/L). An urgent CT pulmonary angiography confirmed bilateral pulmonary embolism and right lower pulmonary infarction, while an ultrasonography showed deep vein thrombosis in her left leg. Further examination revealed that protein S activity was less than 10%. The proband's second sister, a 12-year-old girl, was admitted to the hospital in December 2020. Her protein S activity was 8% and an ultrasonography showed deep vein thrombosis in her right leg. The protein S activity of the proband's father and mother were 36% and 26%, respectively. Trio-whole-exome sequencing detected compound heterozygous PROS1 gene variants (c.-168C>T and c.200A>C (p.E67A)) for the proband and her second sister, that were inherited from her father and mother, respectively. The proband's third sister's protein S activity was 28%; she and the proband's grandfather both carried c.200A>C (p.E67A) variants. The proband and her younger sister were treated with rivaroxaban and responded well during the 3-month follow-up. A total of 1 Chinese report in literature and 18 English literature were retrieved and 14 patients with protein S deficiency caused by homozygous or complex heterozygous variants of PROS1 gene were enrolled, including 8 male and 6 female patients. The ages ranged from 4 days to 35 years. Three patients experienced fulminant purpura or severe intracranial hemorrhage in early neonatal-period, while the remaining 11 patients developed venous thromboembolism in adolescence. Protein S activity was examined in 11 patients, and all showed less than 10% of activity. Missense variants was the most common type of gene variants. Conclusions: For children with pulmonary embolism, if there are no clear risk factors for thrombosis, hereditary protein S deficiency should be considered, and protein S activity should be examined before oral anticoagulant drugs. If protein S activity is less than 10%, protein S deficiency caused by homozygous or complex heterozygous variants should be considered.

[Long-term rituximab treatment of refractory idiopathic inflammatory myopathy: A report of 3 cases].

Idiopathic inflammatory myopathies are a group of rare but serious diseases. The treatment of refractory idiopathic inflammatory myopathy is always challenging, especially in children. Three cases of refractory idiopathic inflammatory myopathy treated by rituximab were reported and discussed with the review of relevant literature. All were female with on-set age of 8 years and 6 months, 11 years and 7 months, 4 years and 2 months old, respectively. All had acute onset, presenting with progressive and severe muscle weakness. All lost ambulation within 1 or 2 months, with difficult swallowing and low voice. Respiratory distress occurred in case 2 after an attack of asphyxia due to an aspiration of sputum, and ventilator support was required for 1 month. Rashes were detected at the initial stage of the disease in cases 2 and 3. Patient 2 showed facial erythematous papules, spreading to her neck and hands. Patient 3 showed purplish eyelids with peri-orbital swelling, generalized edema involving all her limbs. Creatine kinase (CK) levels were markedly elevated in all the patients, ranging from 6 000 IU/L to 28 819 IU/L. Anti-SRP antibody was identified in cases 1, and anti-NXP2 antibodies were confirmed in cases 2 and 3. MRI of both thighs in all the patients showed profound muscle and fascial edema. Muscle pathology of patient 1 showed prominent fiber variation and endomysial fibrosis, with overexpression of MHC-Ⅰ. While muscle pathology in patients 2 and 3 showed scattered fiber necrosis, regeneration, endomysial edema without inflammatory cell infiltration. All the patients were diagnosed with idiopathic inflammatory myopathy and failed to the initial treatment including adequate glucocorticoids and high-dose immunoglobulin therapy. Other immunosuppressants (methotrexate, cyclophosphamide) were also tried in cases 2 and 3 with poor response. Then all the patients were treated with rituximab combined with glucocorticoids. Patient 1 regained normal strength and discontinued rituximab at the end of her last follow-up (2 years and 7 mouths). Though calcinosis developed during the follow-up period, significant improvement was noticed in cases 2 and 3 (both regained the ability to walk independently) at the end of their last follow-up after 2 years and 8 months, 3 years and 2 months respectively. Long-term rituximab therapy may improve the prognosis of refractory idiopathic inflammatory myopathy, especially with positive anti-SRP and anti-NXP2 antibodies.

Muscone alleviates myocardial ischemia-reperfusion injury via inhibition of oxidative stress and enhancement of SIRT3.

This study aimed at probing into the function of muscone in ameliorating myocardial ischemiareperfusion (I/R) injury and exploring the underlying mechanism. To analyze the function of muscone, H9c2 cardiomyocytes were treated with hypoxia/reoxygenation (H/R) and Sprague-Dawley (SD) rats were treated with left anterior descending (LAD) of the coronary artery ligation for 30 min and reperfusion for 2 h to induce myocardial I/R injury. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot were used to detect the expression of SIRT3. MTT assay and TUNEL assay were performed to investigate H9c2 viability and apoptosis, respectively. ELISA was employed to determine the expressions of inflammatory cytokines TNF-α, IL-6 and IL-1ß, and myocardial injury markers CK and LDH. Oxidative stress markers MDA and SOD, and ROS expression levels were also detected. SIRT3 inhibitor 3-TYP was used to further confirm whether muscone worked via the augmentation of SIRT3. Herein, we found that muscone significantly inhibited inflammation and oxidative stress in H9c2 cardiomyocytes in a dose-dependent manner. H9c2 viability was promoted by muscone while apoptosis was inhibited. In SD rats, pre-treatment of muscone alleviated I/R injury-induced cardiac function dysregulation and left ventricle remolding. Furthermore, muscone increased SIRT3 expression at both mRNA and protein levels. With 3-TYP inhibiting SIRT3, the protective effects of muscone in H9c2 cardiomyocytes and SD rats were all significantly alleviated. In summary, muscone can attenuate inflammation, oxidative stress and cardiomyocytes injury in H9c2 cells treated with H/R and alleviate myocardial I/R injury of SD rats, which are dependent on SIRT3.

MiR-638 regulates gastric cardia adenocarcinoma cell proliferation, apoptosis, migration and invasion by targeting MACC1.

Gastric cardia adenocarcinoma (GCA) is one of the most common types of cancer and the incidence is increasing globally. MicroRNAs (miRNAs) have been reported to play critical roles in the progression of GCA. However, the exact role of miR-638 in GCA and its underlying mechanism remain largely unknown. The expression levels of miR-638 and metastasis-associated in colon cancer 1 (MACC1) were measured by quantitative real-time polymerase chain reaction (qRT-PCR). Cell proliferation, apoptosis, migration and invasion were detected by Cell Counting Kit-8 (CCK-8) assay, flow cytometry and transwell assay, respectively. Western blot analysis was performed to determine the protein levels of cleaved-caspase 3 (C-caspase 3) and MACC1. The possible binding sites of miR-638 and MACC1 were predicted by TargetScan online software and confirmed by dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. A xenograft model was established to investigate the roles of MACC1 in GCA in vivo. The expression of miR-638 was evidently reduced and MACC1 expression was obviously enhanced in GCA tissues and cells. Overexpression of miR-638 or knockdown of MACC1 inhibited cell proliferation, migration and invasion but increased apoptosis in GCA cells. Moreover, MACC1 was a direct target of miR-638 and its upregulation attenuated the inhibitory effect of miR-638 overexpression on the progression of GCA. In addition, overexpression of miR-638 significantly decreased tumor growth by downregulating MACCI in vivo. In conclusion, miR-638 overexpression suppressed cell proliferation, migration and invasion but induced cell apoptosis by targeting MACC1 in GCA cells, providing a potential therapeutic strategy for the treatment of GCA.

Lithium recovery with LiTi2O4 ion-sieves.

A feasibility study for the recovery of lithium from salt water with the protonated lithium titanium oxide ion-sieves was carried out in this work. Lithium ions (Li+) in LiTi2O4 having a similar ion density with H+ allow repeated exchanges and regeneration with high selectivity. By Li7 magic angle spinning solid-state magnetic resonance, it is apparent that chemical structure of lithium in the ion-sieves is not perturbed during the repeated Li+/H+ exchange processes. As the dissolution of titanium is negligible (<0.1%), the secondary contamination during the capture process can be minimized. The ion-sieves exhibit lithium capture capacities of up to 9.5mg/g during the repeated Li+/H+ exchanges with H0.23Li0.77Ti2O4/LiTi2O4 for 24h, and the captured Li+ may be recovered in the form of Li2CO3. Accordingly, the lithium capture method developed in this work could be integrated with current desalination processes for valuable lithium recovery.

[Peripheral nerve injury in LAMA2-related congenital muscular dystrophy patients].

Objective: To explore the injury pattern and features of peripheral nerve in congenital muscular dystrophy patients caused by LAMA2 gene mutation. Method: Seventeen patients genetically or molecular pathologically diagnosed as LAMA2-related congenital muscular dystrophy were recruited in Peking University First Hospital between 2002 and 2015. All the patients received nerve conduction velocity (NCV) and needle electromyography tests. Clinical and laboratory examination data of the patients was retrospectively analyzed. The correlation between the NCV and disease course was determined by Pearson correlation analysis. Additionally, one patient underwent a sural nerve biopsy. Result: Among these 17 identified patients (13 male and 4 female), all of them were diagnosed as congenital muscular dystrophy, and all of them underwent electrophysiological examination at ages between 1 month to 6 years. Electromyogram indicated seventeen patients of myogenic damage, of whom 10 cases were complicated with reduced NCV. Twenty-six of 95 analyzed nerves showed NCV slower than the normal average of contemporary in 17%-47%. Correlation analysis between NCV and the disease course indicated that NCV of median nerves, ulnar nerves, tibial nerves and common peroneal nerves were negatively associated with the disease course (r=-0.737, -0.771, -0.540 and -0.682, respectively; all P<0.05). Sural nerve biopsy revealed peripheral neuropathy changes of myelin. Conclusion: There is peripheral nerve injury in LAMA2-related muscular dystrophy patients. It mainly manifests as demyelinating lesions. Moreover, the NCV of peripheral nerve will decrease with the increase of the course of the disease.

[Correlation between thigh muscle magnetic resonance imaging findings and clinical features of congenital muscular dystrophies: a preliminary study].

Objective: To analyze the clinical and magnetic resonance imaging (MRI) features of congenital muscular dystrophy (CMD) to improve the diagnostic level. Method: Clinical manifestations and thigh muscle MRI results of 8 cases of CMD diagnosed on genetic level from April 2013 to November 2015 were investigated. MRI was performed on the thigh muscles of all cases. Fatty infiltration of different muscles described in T1WI was graded to evaluate. Clinical symptoms and signs, as well as muscle MRI features were analyzed by statistical description. Result: Among these 8 cases, 2 cases were diagnosed with Ullrich congenital muscular dystrophy (UCMD), 1 case had rigid spine with muscular dystrophy type 1 (RSMD1), 1 case had LMNA related muscular dystrophy (L-CMD), 1 case had congenital muscular dystrophy 1C (MDC1C) and 3 cases had congenital muscular dystrophy 1A (MDC1A), with 4 were males and 4 females, aged from 0.9 year to 4.8 years (median age was 2.2 years). All of these 8 cases presented with muscle weakness and hypotonia from birth to within the first six months, together with delayed motor development and joint contractures. Some cases had spinal deformity or skin changes. Various degrees of fatty infiltration in gluteus maximus and thigh muscles were shown in all of the cases, and differences among CMD subtypes in the form of fatty infiltration were detected; muscle edema was present in 5 cases, and muscle atrophy in 7 cases. However, none of them has muscle hypertrophy. Semimembranous muscle absence was detected in 1 case. Conclusion: The clinical manifestations and thigh muscle MRI findings of CMD have some features, and vary in certain CMD subtypes. MRI examination combined with clinical features may provide useful information to select appropriate genetic or other diagnostic techniques, which may help clinicians to make accurate diagnosis.

Cadmium directly induced mitochondrial dysfunction of human embryonic kidney cells.

Cadmium (Cd) is the major component of polluted environment, which has numerous undesirable effects on health. Cd could induce apoptosis of HEK293 cells, and the mitochondria may play a key role. However, the mode of action is unclear. In the present study, we aimed to evaluate the ability of the Cd to induce dysfunction of mitochondria. We examined the effect of cadmium chloride (1, 5 and 10 µM) on mitochondrial membrane permeability and potential as well as oxidative stress markers in mitochondria isolated from HEK293 cells. We found that Cd could directly increase in permeability and decrease in membrane potential of mitochondria, even resulted in mitochondrial swelling, and that Cd could inhibit the activities of ATPase, lactate dehydrogenase (LDH), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), enhanced the levels of reactive oxygen species (ROS) and lipid peroxidation (LPO). On the whole, the results show that Cd can directly lead to mitochondrial dysfunction of HEK293 cells, including increased permeability, inhibiting respiration and evoking oxidative stress. Thus, for the first time, this paper makes an overall analysis of Cd-induced changes of structure and function of isolated mitochondria. Our findings may also have general implications in Cd-induced apoptosis by mitochondria pathway.

Systematic analysis of missed extremity fractures in emergency radiology.

PURPOSE: To systematically analyze fractures in the extremities that were missed in the initial radiological report, primarily on plain radiographs, in the emergency department (ED). MATERIAL AND METHODS: From January 2003 to June 2004, 2407 new patients were confirmed to have fractures in the extremities in the ED. A total of 3081 fractures were confirmed. In the initial radiological reports, 115 fractures in 108 patients were missed. One musculoskeletal radiologist and one emergency radiologist independently carried out a second review of these images. The easily missed fracture sites were recorded. The possible reasons for misinterpretation were determined by consensus. RESULTS: The most frequent location for missed fractures, expressed as a percentage of all fractures in the same location, was the foot (7.6%), followed by the knee (6.3%), elbow (6.0%), hand (5.4%), wrist (4.1%), hip (3.9%), ankle (2.8%), and shoulder (1.9%). The average percentage for all missed fractures was 3.7%. On the second review, 70% of the initially missed fractures were identified. Analysis of the possible reasons for missed fractures showed the most common reason was subtlety of the fracture. CONCLUSION: The overall percentage of missed fractures in the extremities was 3.7%. Only 33% of the initially missed fractures were attributed to radiographically imperceptible lesions. Adequate training for physicians and radiologists in the ED may reduce the rate of missed fractures.

Percutaneous vertebroplasty for patients with osteoporosis: a one-year follow-up.

PURPOSE: To evaluate changes in height and wedge angle of treated vertebral bodies and kyphosis angle 1 year after vertebroplasty. MATERIAL AND METHODS: We reviewed radiographs of 95 vertebral bodies treated with vertebroplasty in 60 patients with osteoporosis. Only vertebral bodies with imaging evidence of a new fracture or avascular necrosis received vertebroplasty. Images were obtained for evaluation before vertebroplasty (B), within 2 weeks after vertebroplasty (T), and after 1 year (T1). RESULTS: The mean wedge angle decreased by 5.4 degrees from B to T1. Mean of the anterior, central, and posterior heights of the fractured bodies increased by 12.6%, 9.6%, and 3.1%, respectively, from B to T1. The kyphosis angle improved by 3.2 degrees initially from B to T, but the improvement later disappeared. In 48% of these patients, a new fracture developed after vertebroplasty, and 63% of the fractures were adjacent to a vertebroplasty-treated vertebral body. CONCLUSION: The increase in height and wedge angle of the vertebral bodies generally lasted at least 1 year. Improvement in kyphosis angles was lost 1 year after vertebroplasty because new fractures occurred in 48% of these patients. Prevention of new fractures after vertebroplasty remains an important task.

Indwelled femoral vein non-cuffed, double-lumen hemodialysis catheter complicated by pulmonary thromboembolism.

Non-cuffed, double-lumen hemodialysis (HD) catheters can be inserted at the bedside in the femoral, internal jugular or subclavian position. The femoral route is less risky, and the incidence of life-threatening complications is lower for femoral cannulation than for internal jugular and subclavian cannulations. However, here we describe a life-threatening complication of an extensive deep vein thrombosis and subsequent pulmonary thromboembolism following femoral cannulation of a double-lumen HD catheter. The possible mechanisms and treatment for this potentially fatal thromboembolic event are discussed in this report.

Characterization of the binding of Photobacterium phosphoreum P-flavin by Vibrio harveyi Luciferase.

The isolated Photobacterium phosphoreum luciferase is associated with a bound flavin designated P-flavin and tentatively identified as 6-(3"-myristic acid)-FMN. Since FMN and myristic acid are products of the normal luciferase reaction, we explored the possibility that P-flavin can also be bound by luciferase from other luminous bacteria and serve as an active site probe. P-flavin has never been detected in Vibrio harveyi cells. We found that the V. harveyi luciferase binds P. phosphoreum P-flavin, at a ratio of 1 P-flavin per luciferase alphabeta dimer, and with concomitant absorption spectral perturbation of P-flavin, fluorescence quenching of P-flavin and luciferase, and activity inhibition of luciferase. Isolated P-flavin can be fully reduced photochemically. V. harveyi luciferase bound the oxidized P-flavin with a K(d) (or K(i) competitively against decanal) of 0.1-0.16 microM, which is three orders of magnitude lower than the K(d) for FMN binding but similar to that of reduced FMN binding. The reduced P-flavin exhibited a K(i) (competitively against the reduced FMN substrate) of 0.16 microM, also similar to the K(d) for reduced FMN. Hence, the covalent attachment of myristic acid to FMN greatly and preferentially enhanced the binding of oxidized P-flavin. The dissociation of P-flavin was slow in comparison with the binding of reduced FMN and decanal substrates. Modification of the alphaCys106 near the active site by N-ethylmaleimide can be retarded by P-flavin. These findings indicate that P-flavin is potentially a superb active site probe for luciferase. We hypothesize that P-flavin is a by-product of luciferase generated by a side reaction which is trivial with the V. harveyi luciferase but significant in the P. phosphoreum luciferase-catalyzed reaction.

Action mechanism of antitubercular isoniazid. Activation by Mycobacterium tuberculosis KatG, isolation, and characterization of inha inhibitor.

Activation of the antitubercular isoniazid (INH) by the Mycobacterium tuberculosis KatG produces an inhibitor for enoyl reductase (InhA). The mechanism for INH activation remains poorly understood, and the inhibitor has never been isolated. We have purified the InhA-inhibitor complex generated in the M. tuberculosis KatG-catalyzed INH activation. The complex exhibited a 278-nm absorption peak and a shoulder around 326 nm with a characteristic A(326)/A(278) ratio of 0.16. The complex was devoid of enoyl reductase activity. The inhibitor noncovalently binds to InhA with a K(d) < 0.4 nM and can be dissociated from denatured InhA for chromatographic isolation. The free inhibitor showed absorption peaks at 326 (epsilon(326) 6900 M(-1) cm(-1)) and 260 nm (epsilon(260) 27,000 M(-1) cm(-1)). The inactive complex can be reconstituted from InhA and the isolated inhibitor. The InhA inhibitor from the KatG-catalyzed INH activation was identical to that from a slow, KatG-independent, Mn(2+)-mediated reaction based on high pressure liquid chromatography analysis and absorption and mass spectral characteristics. By monitoring the formation of the InhA-inhibitor complex, we have found that manganese is not essential to the INH activation by M. tuberculosis KatG. Furthermore, the formation of the InhA inhibitor in the KatG reaction was independent of InhA.

Stability and peptide binding specificity of Btk SH2 domain: molecular basis for X-linked agammaglobulinemia.

X-linked agammaglobulinemia (XLA) is caused by mutations in the Bruton's tyrosine kinase (Btk). The absence of functional Btk leads to failure of B-cell development that incapacitates antibody production in XLA patients leading to recurrent bacterial infections. Btk SH2 domain is essential for phospholipase C-gamma phosphorylation, and mutations in this domain were shown to cause XLA. Recently, the B-cell linker protein (BLNK) was found to interact with the SH2 domain of Btk, and this association is required for the activation of phospholipase C-gamma. However, the molecular basis for the interaction between the Btk SH2 domain and BLNK and the cause of XLA remain unclear. To understand the role of Btk in B-cell development, we have determined the stability and peptide binding affinity of the Btk SH2 domain. Our results indicate that both the structure and stability of Btk SH2 domain closely resemble with other SH2 domains, and it binds with phosphopeptides in the order pYEEI > pYDEP > pYMEM > pYLDL > pYIIP. We expressed the R288Q, R288W, L295P, R307G, R307T, Y334S, Y361C, L369F, and 1370M mutants of the Btk SH2 domain identified from XLA patients and measured their binding affinity with the phosphopeptides. Our studies revealed that mutation of R288 and R307 located in the phosphotyrosine binding site resulted in a more than 200-fold decrease in the peptide binding compared to L295, Y334, Y361, L369, and 1370 mutations in the pY + 3 hydrophobic binding pocket (approximately 3- to 17-folds). Furthermore, mutation of the Tyr residue at the betaD5 position reverses the binding order of Btk SH2 domain to pYIIP > pYLDL > pYDEP > pYMEM > pYEEI. This altered binding behavior of mutant Btk SH2 domain likely leads to XLA.