[Analysis of 8 cases of asparaginase related cerebral venous sinus thrombosis in children].

Objective: To summarize the clinical features, treatment and prognosis of asparaginase (ASP) related cerebral venous sinus thrombosis (CVST). Methods: Clinical profiles including age, sex, first symptoms, coagulation function, imaging findings, ASP type, treatment and prognosis of eight acute lymphoblastic leukemia (ALL) or lymphoblastic lymphoma (LBL) children with ASP related CVST at the Department of Pediatrics, First Affiliated Hospital of Zhengzhou University from November 2016 to October 2021 were analyzed retrospectively. Results: Eight CVST children were all male, including 6 ALL and 2 LBL, with the onset age ranged from 5 to 15 years, 6 cases occurred in the stage of first induction remission, and the initial symptom were mainly epileptic seizures (7 cases). Magnetic resonance imaging combined magnetic resonance venography (MRV) showed the most common site of venous sinus enlargement was superior sagittal sinus (8 cases). Secondary cerebral hemorrhage was found in 5 cases. D-dimer elevated on the day of onset in all cases. Three patients were treated with intravascular mechanical thrombectomy and thrombolysis combined with anticoagulant therapy, 3 patients were treated with continuous anticoagulant therapy only, 2 patients were not treated with anticoagulant therapy. MRV follow-up for 3 months showed that the thrombi in patients were almost completely absorbed except in 2 patients who were not treated with anticoagulant therapy. Thrombolysis combined with anticoagulant therapy was the fastest way for thrombosis absorption. Among 8 patients, 1 died of early recurrence of ALL, and 7 patients accepted further asparaginase and no CVST recurrence or progression was found. There were no sequelae of nervous system except 1 patient with left upper limb muscle strength impairment. Conclusions: ASP related CVST is more common in older male children and the prognosis is good. ASP related CVST occurred mostly in the stage of first induction remission, and most initial manifestation is epileptic seizure. The superior sagittal region is a common site of thrombus, magnetic resonance imaging combined with MRV is helpful for accurately diagnosis. Timely anticoagulant treatment can improve the prognosis, and mechanical thrombectomy and thrombolysis can quickly recanalize the vessel.

Impaired glucose tolerance in first-episode drug-naïve patients with schizophrenia: relationships with clinical phenotypes and cognitive deficits.

BACKGROUND: Schizophrenia patients have a higher prevalence of type 2 diabetes mellitus with impaired glucose tolerance (IGT) than normals. We examined the relationship between IGT and clinical phenotypes or cognitive deficits in first-episode, drug-naïve (FEDN) Han Chinese patients with schizophrenia. METHOD: A total of 175 in-patients were compared with 31 healthy controls on anthropometric measures and fasting plasma levels of glucose, insulin and lipids. They were also compared using a 75 g oral glucose tolerance test and the homeostasis model assessment of insulin resistance (HOMA-IR). Neurocognitive functioning was assessed using the MATRICS Consensus Cognitive Battery (MCCB). Patient psychopathology was assessed using the Positive and Negative Syndrome Scale (PANSS). RESULTS: Of the patients, 24.5% had IGT compared with none of the controls, and they also had significantly higher levels of fasting blood glucose and 2-h glucose after an oral glucose load, and were more insulin resistant. Compared with those patients with normal glucose tolerance, the IGT patients were older, had a later age of onset, higher waist or hip circumference and body mass index, higher levels of low-density lipoprotein and triglycerides and higher insulin resistance. Furthermore, IGT patients had higher PANSS total and negative symptom subscale scores, but no greater cognitive impairment except on the emotional intelligence index of the MCCB. CONCLUSIONS: IGT occurs with greater frequency in FEDN schizophrenia, and shows association with demographic and anthropometric parameters, as well as with clinical symptoms but minimally with cognitive impairment during the early course of the disorder.

The Chinese First-Episode Schizophrenia Trial: background and study design.

Schizophrenia is a complex illness with unknown aetiology and pathogenesis. Currently, a considerable number of patients with schizophrenia do not receive standardised and systematic treatment in China. In the past years, many controlled trials have been conducted in chronic schizophrenia. In contrast, research on first-episode schizophrenia is lacking. This paper describes the background and design of the Chinese First-Episode Schizophrenia Trial project--a multicentre, randomised, open-label clinical trial. A total of 600 first-episode schizophrenia patients were randomly divided into 3 groups and treated with risperidone, aripiprazole, and olanzapine for 1 year. During the study period, only 1 medication change of the 3 antipsychotic medications was allowed.

Chimeric small subunit inhibitors of mammalian ribonucleotide reductase: a dual function for the R2 C-terminus?

Here we report on the formation and activity of complexes between the large subunit (mR1) dimer of mouse ribonucleotide reductase (mRR) and small subunit chimeric dimers (cR2) derived from Escherichia coli and mouse small subunits. cR2 subunits were constructed by substituting mouse C-terminal gene sequences, coding for either 7 or 33 amino acid residues, for the corresponding E.coli R2 (eR2) sequences, with the remainder of the gene corresponding to eR2. The purified cR2s contained the micro-oxo bridged diferric center and tyrosine radical necessary for reductase activity, although the radical signal was broadened compared with wild-type eR2. Neither chimera formed an active complex with mR1, but each was a competitive inhibitor, with respect to mR2, of mRR activity. The inhibition constants for both chimeras were similar, and were sevenfold higher than the dissociation constant of mR2 dimer to mR1 dimer (0.24 +/- 0.02 microM). Analysis of inhibition data showed that chimeric R2 subunits bind to mammalian R1 with a 1:1 (R1:R2) stoichiometry and permit the inference that both C-termini in a cR2 dimer bind to the two sites per mR1 dimer. The lack of enzymatic activity in the mR1-cR2 complex is attributed to perturbation or elimination of interactions linking the tyrosine radical/dinuclear iron center and the C-terminus within R2.

R2 C-terminal peptide inhibition of mammalian and yeast ribonucleotide reductase.

Eucaryotic ribonucleotide reductases (RR) catalyze the reduction of ribonucleoside diphosphates to 2'-deoxyribonucleoside diphosphates. Each has an R1(2)R2(2) quaternary structure with each subunit playing a critical role in catalysis. Separation of the subunits results in loss of activity. Previous studies have demonstrated that peptides corresponding to the C-terminus of R2 disrupt subunit association by competion with R2 and have potential usefulness as therapeutics. Extensive structure-function studies have been carried out on peptide inhibition of herpes simplex RR in an effort to develop antiviral agents based on the observation that the herpes simplex R2 C-terminus, YAGAVVNDL, is quite different from the corresponding mammalian sequence. In this work we report a detailed structure-function analysis of peptide inhibition of mammalian and, to a more limited extent, Saccharomyces cerevisiae RRs. Our results for mammalian RR support the following conclusions with regard to the effect of substitution on inhibitory potency: (a) the N-acetylated R2 C-terminal heptapeptide N-AcPhe384Thr385Leu386Asp387Ala388Asp389Phe390 (N-AcF7TLDADF1) is the minimal core peptide length required; deletion of the N-terminus or of middle positions (resulting in penta- and hexapeptides) results in large losses in inhibitory potency; (b) a free carboxylate is required on the C-terminal Phe; (c) Phe is strongly preferred to Leu in positions 1 and 7 and a bulky aliphatic group is preferred in position 5; (d) neither negative charge in positions 2 or 4 nor a polar side chain in position 6 are required for peptide binding, contrary to what evolutionary patterns in the R2 C-terminus of RR would suggest. S. cerevisiae RR displays a similar length dependence on the corresponding N-acetylated R2 C-terminal heptapeptide, N-AcFTFNEDF. This peptide has a 4-fold higher inhibitory potency toward S. cerevisiae RR than toward mammalian RR. Such selectivity raises the possibility that peptide analogs related to R2 C-termini can be developed as therapeutic agents even against organisms having R2 C-terminal sequences similar to that of mammalian RR.

Cloning, sequence determination, and regulation of the ribonucleotide reductase subunits from Plasmodium falciparum: a target for antimalarial therapy.

Malaria remains a leading cause of morbidity and mortality worldwide, accounting for more than one million deaths annually. We have focused on the reduction of ribonucleotides to 2'-deoxyribonucleotides, catalyzed by ribonucleotide reductase, which represents the rate-determining step in DNA replication as a target for antimalarial agents. We report the full-length DNA sequence corresponding to the large (PfR1) and small (PfR2) subunits of Plasmodium falciparum ribonucleotide reductase. The small subunit (PfR2) contains the major catalytic motif consisting of a tyrosyl radical and a dinuclear Fe site. Whereas PfR2 shares 59% amino acid identity with human R2, a striking sequence divergence between human R2 and PfR2 at the C terminus may provide a selective target for inhibition of the malarial enzyme. A synthetic oligopeptide corresponding to the C-terminal 7 residues of PfR2 inhibits mammalian ribonucleotide reductase at concentrations approximately 10-fold higher than that predicted to inhibit malarial R2. The gene encoding the large subunit (PfR1) contains a single intron. The cysteines thought to be involved in the reduction mechanism are conserved. In contrast to mammalian ribonucleotide reductase, the genes for PfR1 and PfR2 are located on the same chromosome and the accumulation of mRNAs for the two subunits follow different temporal patterns during the cell cycle.

The carboxyl terminus heptapeptide of the R2 subunit of mammalian ribonucleotide reductase inhibits enzyme activity and can be used to purify the R1 subunit.

The heptapeptide, FTLDADF, identical in sequence to the last seven amino acid residues of the carboxyl terminus of the R2 subunit of mouse ribonucleotide reductase (RR), and its N alpha-acetyl derivative both inhibit calf thymus RR. The N alpha-acetyl derivative is considerably more potent, displaying a K1 of 20 microM. The same K1 was found for N-AcFTLDADF inhibition of a reconstituted ribonucleotide reductase from calf thymus R1 and mouse R2, indicating that the C-termini of calf R2 and mouse R2 might be identical. Our results, taken together with previous results of others on inhibition of viral RR, suggest that inhibition of RRs by peptides mimicking the C-terminus of R2 may be a general phenomenon. In addition, we have shown that an affinity column, FTLDADF-Sepharose 4B, can be used to prepare approximately 95% pure calf thymus R1, devoid of contamination with R2, in a very simple procedure that should be generally applicable to R1 purification from many sources.

The nature of the inhibition of 4,7-dioxobenzothiazole derivatives on mitochondrial ubiquinol-cytochrome c reductase.

To investigate the inhibitory action and binding site of a quinone-like molecule, 5-undecyl-6-hydroxy-4,7-dioxobenzothiazole (UHDBT), a series of 4,7-dioxobenzothiazole derivatives were synthesized and their inhibitory efficiencies studied. Replacing the 6-hydroxyl or 2-hydrogen of UHDBT with a bromo or a methoxy group causes only a slight decrease in inhibitory efficiency, indicating that the 6-hydroxyl or the 2-hydrogen of UHDBT is not a structural requirement for inhibition. 5-Undecyl-6-bromo (or methoxy)-4,7-dioxobenzothiazole shows a pH-dependent inhibition similar to that observed with UHDBT, suggesting that the pH dependence is due to the presence of a dissociable group in the protein complex and not to the deprotonation of the hydroxyl group of the inhibitor. Replacing the 6-hydroxyl group with an azido group causes changes similar to those observed with UHDBT; the inhibition is accompanied by alteration of the epr characteristics of reduced iron-sulfur protein in ubiquinol-cytochrome c reductase. The extent of inhibition is not changed upon illumination of the treated reductase. When the photolyzed, 6-azido-5-(1',2'-[3H] undecyl)-4,7-dioxobenzothiazole [( 3H]6-azido-UDBT)-treated reductase is subjected to organic solvent extraction, no radioactivity is found in the reductase protein. Rather, the radioactivity is located in the phospholipid fraction. A [3H]azido-UDBT-cardiolipin adduct, identified after separation of the phospholipid fraction by high performance liquid chromatography, has 6-azido-UDBT linked to an acyl group, not to the head group of the cardiolipin molecule. These results suggest that inhibition by UHDBT is due to perturbation of specific cardiolipin molecules in ubiquinol-cytochrome c reductase. Since UHDBT and 6-azido-UDBT also inhibit the ubiquinol-cytochrome c reductase activity of delipidated reductase (10% of the original lipid remaining) assayed after reconstitution with ubiquinone and phospholipid, and the [3H]azido-UDBT-cardiolipin adduct is also found in the delipidated reductase, the UHDBT-perturbed cardiolipin molecule is structurally indispensable to reductase and it tightly bound to the reductase protein, most likely the quinone binding proteins.

Phospholipid-dependent interaction between dibromothymoquinone and iron-sulfur protein in mitochondrial ubiquinol-cytochrome c reductase.

Dibromothymoquinone (DBMIB) inhibits antimycin A-sensitive ubiquinol-cytochrome c reductase activity; the maximal inhibition is 90%. DBMIB alters the EPR spectra of reduced iron-sulfur protein in intact ubiquinol-cytochrome c reductase. The maximal spectral change occurs with 60 mol inhibitor per mol cytochrome c1 in the reductase. DBMIB causes little alteration in the EPR characteristics of iron-sulfur protein when ubiquinol-cytochrome c reductase is delipidated. When delipidated ubiquinol-cytochrome c reductase is replenished with phospholipid, the effect of DBMIB reappears. However, when DBMIB is added to delipidated protein prior to replenishment with phospholipid, very little spectral alteration is observed. DBMIB does not alter the EPR spectra of purified iron-sulfur protein, with or without phospholipid in the preparation. Reduced DBMIB does not alter the EPR characteristics of iron-sulfur protein in intact or delipidated ubiquinol-cytochrome c reductase. Cysteine and other thiol compounds can reverse the spectral alternation caused by DBMIB. This reversal probably results from the reduction of DBMIB.

Use of an azido-ubiquinone derivative to identify subunit I as the ubiquinol binding site of the cytochrome d terminal oxidase complex of Escherichia coli.

The radiolabeled, photoreactive azido-ubiquinone derivative (azido-Q), 3-azido-2-methyl-5-methoxy-6-(3,7-dimethyl-[3H]octyl)- 1,4-benzoquinone, was used to investigate the active site of ubiquinol oxidase activity of the cytochrome d complex, a two-subunit terminal oxidase of Escherichia coli. The azido-Q, when reduced by dithioerythritol, was shown to support enzymatic oxygen consumption by the cytochrome d complex that was 8% of the rate observed with ubiquinol-1. This observation provided the rationale behind further studies of the possible photoinactivation and labeling of the active site by this azido-Q. Ten min of photolysis of the purified cytochrome d complex in the presence of the azido-Q resulted in a 60% loss of the ubiquinol-1 oxidase activity. Uptake of the radiolabeled azido-Q by the cytochrome d complex was correlated to the photoinactivation of the ubiquinol-1 oxidase activity. Both increased linearly during the first 4 min of photolysis and reached 90% of the maximum within 10 min. Photolysis times longer than 10 min resulted in no increase in the maximum of 2 mol of azido-Q incorporated per mol of enzyme. The rate of azido-Q uptake by subunit I, but not subunit II, correlated well with the rate of loss of ubiquinol oxidase activity. Use of ubiquinol-0, which is not oxidized by the enzyme, to competitively inhibit radiolabeling of nonspecific binding sites, resulted in a significant decrease (42%) of azido-Q labeling of subunit II while it did not affect the labeling of subunit I. After photolysis for 4 min, the ratio of radiolabeled azido-Q in subunits I to II of the complex was 4.3 to 1.0. These observations support the conclusion that the ubiquinol substrate binding site is located on subunit I of the cytochrome d complex.

Identification of ubiquinone-binding proteins in yeast mitochondrial ubiquinol-cytochrome c reductase using an azido-ubiquinone derivative.

An azido-ubiquinone derivative, 3-azido-2-methyl-5-methoxy-6-(3,7-dimethyloctyl)-1,4-benzoquinone, was used to study the ubiquinone-protein interaction and to identify the ubiquinone-binding proteins in yeast mitochondrial ubiquinone-cytochrome c reductase. The phospholipids and Q6 in purified reductase were removed by repeated ammonium sulfate precipitation in the presence of 0.5% sodium cholate. The resulting phospholipid- and ubiquinone-depleted reductase shows no enzymatic activity; activity can be completely restored by the addition of phospholipids and Q6 or Q2. The ubiquinone- and phospholipid-replenished ubiquinonol-cytochrome c reductase is also fully active upon reconstituting with bovine succinate-ubiquinone reductase to form succinate-cytochrome c reductase. When an azido-ubiquinone derivative was added to the ubiquinone and phospholipid-depleted reductase in the dark, followed by the addition of phospholipids, partial reconstitutive activity was restored, while full ubiquinol-cytochrome c reductase activity was observed when Q2H2 was used as substrate in the assay mixture. Apparently, the large amount of Q2H2 present in the assay mixture displaces the azido-ubiquinone in the system. Photolysis of the azido-Q-treated reductase with long-wavelength ultraviolet light abolishes about 70% of both the restored reconstitutive activity and Q2H2-cytochrome c reductase activity. The activity loss is directly proportional to the covalent binding of [3H]azido-ubiquinone to the reductase protein. When the photolyzed, [3H]azido-ubiquinone-treated sample was subjected to SDS-polyacrylamide gel electrophoresis followed by analysis of the distribution of radioactivity among the subunits, the cytochrome b protein and a protein with an apparent molecular weight of 14 000 were heavily labeled. The amount of radioactive labeling in both these proteins was affected by the presence of phospholipids.

Interaction and identification of ubiquinone-binding proteins in ubiquinol-cytochrome c reductase by azido-ubiquinone derivatives.

Various azido-ubiquinone derivatives were synthesized and characterized. 3-Azido-2-methyl-5-methoxy-6-(3,7-dimethyloctyl)-1,4-benzoquinone was found to be suitable for the study of specific interaction between ubiquinone (Q) and protein. It was synthesized with high specific radioactivity and used to identify the Q-binding proteins in purified ubiquinol-cytochrome c reductase. This azido-Q derivative showed partial efficiency in restoring activity to the Q- and phospholipids-depleted ubiquinol-cytochrome c reductase in the absence of light. Azido-Q derivative treated samples, however, became completely inactivated upon photolysis, and the inactivation was not reversed by addition of Q derivatives. The redox state of the azido-Q derivative has little effect on the Q-binding affinity. Two protein subunits with Mr = 37,000 and 17,000 were found to be heavily labeled when depleted ubiquinol-cytochrome c reductase was treated with [3H] azido-Q derivative followed by photolysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amount of radioactive labeling of the Mr = 17,000 protein was proportional to the degree of inactivation and affected by the presence of phospholipids. The radioactive labeling of the Mr = 37,000 protein subunit, however, showed no correlation with degree of inactivation and was not affected by phospholipids. Since the radiolabeling at the Mr = 17,000 protein subunit was affected by phospholipids and correlated with the enzymatic activity, this subunit is probably the Q-binding protein in this enzyme complex (QPc). The inhibition of enzymatic activity by n-heptyl-4-hydroxyquinoline-N-oxide was easily reversed by addition of the azido-Q derivative. The distribution of radioactivity among the subunits of ubiquinol-cytochrome c reductase was not affected by the presence of antimycin A, 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole or n-heptyl-4-hydroxyquinoline-N-oxide, suggesting that the binding site(s) of these inhibitors are not the Q-binding site.