The rapid reversible inhibition of band 3-mediated inorganic anion transport by 4,4′-diisothiocyanodihydrostilbene-2,2′-disulfonate (H
2DIDS) turns slowly into irreversible inhibition. This is due to ...covalent bond formation of the two isothiocyanate groups of the inhibitor with two lysine residues on band 3, called Lys
a and Lys
b. In the red cell membrane, the p
K value of Lys
a is about 2.5 p
K units lower than the p
K value of Lys
b. Hence the susceptibility of Lys
a to irreversible modification by H
2DIDS far exceeds the susceptibility to Lys
b. In the present paper, we have expressed in
Xenopus oocytes cRNA's derived from cDNA clones encoding wild-type mouse band 3 and mouse band 3 in which Lys
a (Lys-558) had been replaced by an Asn residue by oligonucleotide-directed mutagenesis. In accord with previous findings, in the oocytes both wild-type and mutated band 3 mediate Cl
− exchange. After determining the uninhibited exchange rate the oocytes were exposed for a fixed length of time to H
2DIDS at a concentration (20 μM) which saturates all H
2DIDS binding sites with reversibly bound H
2DIDS (
K
I = 0.3
μM and 1.1.
μM, respectively, for wild-type and mutant). Exposure was terminated by washing with a medium in which H
2DIDS was replaced by bovine serum albumin to remove free and reversibly bound H
2DIDS from the extracellular phase. Subsequent measurements of Cl
− efflux yielded a measure for the irreversible inhibition that persisted. Since the transition from reversible to irreversible H
2DIDS binding was found to follow first-order kinetics it was possible to calculate rate constants. From the pH dependence of the rate constants, p
K values were calculated. These calculations could be made since in the wild-type, in which Lys
a and Lys
b are present, the exposure to H
2DIDS could be confined to a pH range in which little if any covalent binding to Lys
b takes place. The data could be represented by a single p
K value of 8.3. In the mutant, Lys
a is missing. Hence, covalent reaction can only take place with Lys
b. Measurements over the appropriate pH range could be described by a single p
K of 10.8. These values are 0.8–0.9 p
K units higher than those previously obtained in experiments with band 3 in the red cell membrane (Kampmann et al. (1982) J. Membr. Biol. 70, 199–216). The difference can be accounted for by the difference of temperature which amounted to 20°C in the present experiments with the oocytes and to 30°C in the previous work with red blood cells. The results suggest that location and function of Lys
a and Lys
b are essentially the same after expression in the lipid bilayer of the red blood cell and the oocyte.
Neuroblastoma is the most common extracranial solid tumour in children. Relapsed or refractory neuroblastoma is associated with a poor outcome. We assessed the combination of irinotecan–temozolomide ...and dasatinib–rapamycin (RIST) in patients with relapsed or refractory neuroblastoma.
The multicentre, open-label, randomised, controlled, phase 2, RIST-rNB-2011 trial recruited from 40 paediatric oncology centres in Germany and Austria. Patients aged 1–25 years with high-risk relapsed (defined as recurrence of all stage IV and MYCN amplification stages, after response to treatment) or refractory (progressive disease during primary treatment) neuroblastoma, with Lansky and Karnofsky performance status at least 50%, were assigned (1:1) to RIST (RIST group) or irinotecan–temozolomide (control group) by block randomisation, stratified by MYCN status. We compared RIST (oral rapamycin loading 3 mg/m2 on day 1, maintenance 1 mg/m2 on days 2–4 and oral dasatinib 2 mg/kg per day for 4 days with 3 days off, followed by intravenous irinotecan 50 mg/m2 per day and oral temozolomide 150 mg/m2 per day for 5 days with 2 days off; one course each of rapamycin–dasatinib and irinotecan–temozolomide for four cycles over 8 weeks, then two courses of rapamycin–dasatinib followed by one course of irinotecan–temozolomide for 12 weeks) with irinotecan–temozolomide alone (with identical dosing as experimental group). The primary endpoint of progression-free survival was analysed in all eligible patients who received at least one course of therapy. The safety population consisted of all patients who received at least one course of therapy and had at least one post-baseline safety assessment. This trial is registered at ClinicalTrials.gov, NCT01467986, and is closed to accrual.
Between Aug 26, 2013, and Sept 21, 2020, 129 patients were randomly assigned to the RIST group (n=63) or control group (n=66). Median age was 5·4 years (IQR 3·7–8·1). 124 patients (78 63% male and 46 37% female) were included in the efficacy analysis. At a median follow-up of 72 months (IQR 31–88), the median progression-free survival was 11 months (95% CI 7–17) in the RIST group and 5 months (2–8) in the control group (hazard ratio 0·62, one-sided 90% CI 0·81; p=0·019). Median progression-free survival in patients with amplified MYCN (n=48) was 6 months (95% CI 4–24) in the RIST group versus 2 months (2–5) in the control group (HR 0·45 95% CI 0·24-0·84, p=0·012); median progression-free survival in patients without amplified MYCN (n=76) was 14 months (95% CI 9–7) in the RIST group versus 8 months (4–15) in the control group (HR 0·84 95% CI 0·51–1·38, p=0·49). The most common grade 3 or worse adverse events were neutropenia (54 81% of 67 patients given RIST vs 49 82% of 60 patients given control), thrombocytopenia (45 67% vs 41 68%), and anaemia (39 58% vs 38 63%). Nine serious treatment-related adverse events were reported (five patients given control and four patients given RIST). There were no treatment-related deaths in the control group and one in the RIST group (multiorgan failure).
RIST-rNB-2011 demonstrated that targeting of MYCN-amplified relapsed or refractory neuroblastoma with a pathway-directed metronomic combination of a multkinase inhibitor and an mTOR inhibitor can improve progression-free survival and overall survival. This exclusive efficacy in MYCN-amplified, relapsed neuroblastoma warrants further investigation in the first-line setting.
Deutsche Krebshilfe.
Wood is one of the most widely used materials for construction projects, in the furniture industry, as well as in the field of cultural property of valuable objects. Terahertz (THz) radiation has ...great potential for non-contact and non-destructive analysis of wooden materials. This article reports a study of the degree of growth of
in wood samples, using THz radiation. The analysis compares the THz results to microscopic examinations of the samples, common beech incubated with
, at different growth times. The results indicate a strong correlation between the parameters extracted from THz measurements and the progress of the fungal infestation, suggesting that THz imaging may be a valuable tool for non-contact inspection of wood.
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