Main Mechanism Of Inactivation Research Articles

Steady-state operation of a biofilter coupled with photocatalytic control of bacterial bioaerosol emissions.

Bioaerosols are emitted during the biological treatment of water, soil, and air pollutants. The elimination of these pollutants has become a priority due to their detrimental effects on human health. Advanced oxidation technologies have been used to control bioaerosol emissions specially to improve indoor air quality. This investigation was focused on evaluating the biofiltration of ethyl acetate vapors in terms of removal efficiency and bioaerosol emission. Also, a continuous photocatalytic process to inactivate bioaerosols emitted from the biofilter was assessed as a post-treatment. The photocatalysis was developed with ZnO and TiO2 immobilized onto Poraver glass beads. Flow cytometry (FC) coupled with fluorochromes was used to characterize and quantify bioaerosol emissions in terms of live, dead, and injured cells. Ethyl acetate removal efficiencies were maintained in a steady state with values of 100% under 60-gm-3h-1 inlet load (IL). Biomass concentration in the biofilter reached values up to 228mgbiomassgperlite-1 at day 56 of operation, but the spontaneous occurrence of predatory mites diminished biomass concentration by 33%. Bioaerosols emitted during the steady-state operation of the biofilter were composed mainly by bacteria (~ 94%) and in a less extent of fungal spores (0.29-6%). The most efficient photocatalytic system comprised TiO2/Poraver with 78% inactivation of bioaerosols during the first 2h of the process, whereas the ZnO/Poraver system showed null activity (~ 0%) of inactivation. FC results show that the main mechanism of inactivation of TiO2/Poraver was cell death.

Inactivation of Escherichia coli K12 in phosphate buffer saline and orange juice by high hydrostatic pressure processing combined with freezing

Synergistic action of high hydrostatic pressure (HHP) and freezing on inactivation of Escherichia coli K12 in phosphate buffered saline (PBS) was investigated by employing response surface methodology. Samples containing E. coli were stored at 4, −24 and −80 °C overnight before they were pressurized. A maximum of 1.83 log reduction of CFU·ml−1 was obtained following a 9-min treatment at 400 MPa and 4 ± 1 °C in samples stored at 4 °C whereas, 5.63 and 6.83 log reductions were obtained in samples frozen at −24 and −80 °C, respectively. Major disruption of E. coli cells observed by scanning electron microscopy and increased amounts of DNA and RNA measured in pressure treated frozen PBS samples indicated that the main mechanism of inactivation in frozen samples was due to cell rapture. The validity of enhanced microbial inactivation by freezing before HHP for a real food system was tested by using orange juice. Pressurization (250 MPa, 15 min) of frozen (−80 °C) orange juice resulted in 4.88, 4.15 and 4.61 log CFU·ml−1 reductions in number of E. coli for the samples having pH 3.2, 4.5 and 5.8, respectively. In the absence of freezing, the same treatment caused only up to 0.42 log reduction in samples having pH 4.5 and 5.8.

Abstract 311: Loss of the tumor suppressor genes CDKN2A/ARF impairs prognosis in adult BCR-ABL1 positive acute lymphoblastic leukemia (ALL)

Abstract Rational: The tumor suppressor genes p16 INK4a /CDKN2A, p14/ARF and p15 INK4b /CDKN2B on 9p21 locus encode critical factors for the regulation of cell cycle and/or apoptosis and are often inactivated in many tumors. Aims: To explore the frequency and size of deletions affecting the 9p21 locus in adult BCR-ABL1-positive ALL, to determine the main mechanism of inactivation and to investigate their prognostic role. Patients and methods: 112 patients were analyzed: 82 (73%) de novo cases, 11 (10%) unpaired relapse cases, 19 (17%) diagnosis-relapse matched samples. The median age was 53 years (range: 18-76) and the median blast percentage was 90% (range, 18-99). Affymetrix single nucleotide polymorphism (SNP) arrays (GeneChip® Human Mapping 250K NspI and Genome-Wide Human SNP 6.0) were used to identify at a high resolution copy number changes on 9p21; PCR amplification and subsequent sequencing were performed in search of mutations; gene-expression profiling (Affymetrix Human Exon 1ST Array) was performed to draw a specific signature associated with deletions. Results: At diagnosis, CDKN2A/ARF and CDKN2B genomic alterations were identified in 29% and 24% of patients, respectively. Deletions were monoallelic in the majority of cases (72%) with a median of 1,012 kb in size (range, 2.8-31,319 kb). In some of them, 43%, the minimal overlapping region of the lost area spanned only the CDKN2A/ARF/CDKN2B gene locus, but more often (57%) the loss was considerable larger and extended sometimes over the entire short arm. Analysis of the genomic status at the relapse showed an almost significant increase in the detection rate of CDKN2A/ARF loss (47%) compared to diagnosis (p = 0.06). The mutation screening performed on each exon of ARF, CDKN2A and CDKN2B genes showed that the 9p21 locus is rarely affected by missense point mutations with only the identification of D146N in the CDKN2A exon 2. Furthermore, 2 mutations were found in the 5’UTR of CDKN2A [UTR A 21965011 (33%), UTR G/A 21965011 (47%), UTR C/T 21964875 (3%)]. Since the prognostic relevance of CDKN2A/ARF deletions is still controversial in literature, we addressed this issue in our study cohort (81 patients evaluable), finding out that deletions in this locus are significantly associated with poor outcome both in terms of disease free-survival (p=0.001) and cumulative incidence of relapse (p=0.001). Conclusions: The inactivation of CDKN2A/ARF is a frequent event in Ph+ ALL. Deletions are frequently acquired at the leukemia progression and work as a poor prognostic marker, impairing disease free-survival and cumulative incidence of relapse. Novel treatment strategies targeting the ARF-MDM2-p53 pathway may be effective in this subset of patients and in vitro studies are ongoing to confirm this hypothesis. Supported by: European LeukemiaNet, AIL, AIRC, FIRB 2006, Programma di Ricerca Regione – Università 2007 – 2009. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 311. doi:10.1158/1538-7445.AM2011-311

The Inactivation of the Tumor Suppressor Genes CDKN2A/ARF by Genomic Deletions Frequently Occurs and Worsens Prognosis In Adult BCR-ABL1 Positive Acute Lymphoblastic Leukemia (ALL) Patients

AbstractAbstract 3136Deletions of the 9p21 chromosomal region are frequent events for the development of a variety of cancers, including solid tumors and hematological malignancies, such as childhood ALL. This region contains the 40-kb region encoding the p16INK4a/CDKN2A (cyclin-dependent kinase inhibitor 2a) tumor suppressor gene and two other related genes, p14/ARF and p15INK4b/CDKN2B, all of which encode critical factors for the regulation of cell cycle and/or apoptosis. In order to explore the frequency and size of deletions affecting the 9p21 locus in adult BCR-ABL1-positive ALL, to determine the main mechanism of inactivation and to investigate the influence on the prognosis, 112 patients were analyzed: 82 (73%) de novo cases, 11 (10%) unpaired relapse cases, 19 (17%) diagnosis-relapse matched samples. The median age was 53 years (range: 18–76) and the median blast percentage was 90% (range, 18–99). Affymetrix single nucleotide polymorphism (SNP) arrays (GeneChip® Human Mapping 250K NspI and Genome-Wide Human SNP 6.0) were used to identify at a high resolution copy number changes on 9p21. PCR amplification and mutation screening of each exon by cloning and subsequent sequencing were also performed. Moreover, gene-expression profiling was performed (Affymetrix Human Exon 1ST Array) to draw a specific signature associated with deletions. At diagnosis, CDKN2A/ARF and CDKN2B genomic alterations were identified in 29% and 24% of patients, respectively. Deletions were monoallelic in the majority of cases (72%) with a median of 1,012 kb in size (range, 2.8–31,319 kb). In some of them, 43%, the minimal overlapping region of the lost area spanned only the CDKN2A/ARF/CDKN2B gene locus, but more often (57%) the loss was considerable larger and extended sometimes over the entire short arm eliminating a large number of genes. In contrast, cases with bi-allelic inactivation were 28% with the majority of deletions (75%) limited to CDKN2A/ARF/CDKN2B genes. FISH analysis was performed using three different BAC clones, but since they overlooked microdeletions we only appreciated a mild fluorescent signal reduction. In order to assess whether 9p21 loss is responsible for progression, the genomic status of this locus was assessed at the time of relapse and an almost significant increase in the detection rate of CDKN2A/ARF loss (47%) compared to diagnosis (p = 0.06) was found. Functionally, deletions led to a strong down-regulation at the transcript level of CDKN2A/ARF (p= 0.0005), as demonstrated by Fluidigm Dynamic Array real-time qPCR assay (Fluidigm Corporation, South San Francisco, CA) which enables to perform TaqMan nano-reactions at high sensitivity. Finally, the mutation screening performed on each exon of ARF, CDKN2A and CDKN2B genes showed that the 9p21 locus is rarely affected by point mutations with only the identification of the missense D146N in the CDKN2A exon 2. Furthermore, 2 mutations were found in the 5′UTR of CDKN2A [UTR A 21965011 (33%), UTR G/A 21965011 (47%), UTR C/T 21964875 (3%)] and a variation, known as SNP was found in the exon 2 (rs3731249). Finally, 2 SNPs were found in the 3′ UTR region of CDKN2A/ARF (rs11515 and rs3088440). The role of the mutations identified in the UTR is not yet defined, but the comparison with the germline material from saliva samples showed that these mutations were inherited. Since the prognostic relevance of CDKN2A/ARF deletions is still controversial in literature, we addressed this issue in our study cohort, finding out that deletions in this locus are significantly associated with poor outcome both in terms of disease free-survival (p=0.003) and cumulative incidence of relapse (p= 0.004). In conclusion, the inactivation of the tumor suppressor genes CDKN2A/ARF is a frequent event in Ph+ ALL. The main mechanism of inactivation is represented by genomic deletions, whereas missense mutations are rare. Deletions are frequently acquired at the leukemia progression and work as a poor prognostic marker, impairing disease free-survival and cumulative incidence of relapse. Novel treatment strategies targeting the ARF-MDM2-p53 pathway may be effective in this subset of patients and in vitro studies are ongoing to confirm this hypothesis. Supported by: European LeukemiaNet, AIL, AIRC, Fondazione Del Monte di Bologna e Ravenna, FIRB 2006, Ateneo RFO grants, Project of integreted program (PIO), Programma di Ricerca Regione – UniversitaÌ€ 2007 – 2009. Disclosures:Baccarani:Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria. Martinelli:Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Pfizer: Consultancy.

The PAX5 gene is frequently rearranged in BCR-ABL1-positive acute lymphoblastic leukemia but is not associated with outcome. A report on behalf of the GIMEMA Acute Leukemia Working Party

Recently, in genome-wide analyses of DNA copy number abnormalities using single nucleotide polymorphism microarrays, genetic alterations targeting PAX5 were identified in over 30% of pediatric patients with acute lymphoblastic leukemia. So far the occurrence of PAX5 alterations and their clinical correlation have not been investigated in adults with BCR-ABL1-positive acute lymphoblastic leukemia. The aim of this study was to characterize the rearrangements on 9p involving PAX5 and their clinical significance in adults with BCR-ABL1-positive acute lymphoblastic leukemia. Eighty-nine adults with de novo BCR-ABL1-positive acute lymphoblastic leukemia were enrolled into institutional (n=15) or GIMEMA (Gruppo Italiano Malattie EMatologiche dell'Adulto) (n=74) clinical trials and, after obtaining informed consent, their genome was analyzed by single nucleotide polymorphism arrays (Affymetrix 250K NspI and SNP 6.0), genomic polymerase chain reaction analysis and re-sequencing. PAX5 genomic deletions were identified in 29 patients (33%) with the extent of deletions ranging from a complete loss of chromosome 9 to the loss of a subset of exons. In contrast to BCR-ABL1-negative acute lymphoblastic leukemia, no point mutations were found, suggesting that deletions are the main mechanism of inactivation of PAX5 in BCR-ABL1-positive acute lymphoblastic leukemia. The deletions were predicted to result in PAX5 haploinsufficiency or expression of PAX5 isoforms with impaired DNA-binding. Deletions of PAX5 were not significantly correlated with overall survival, disease-free survival or cumulative incidence of relapse, suggesting that PAX5 deletions are not associated with outcome. PAX5 deletions are frequent in adult BCR-ABL1-positive acute lymphoblastic leukemia and are not associated with a poor outcome.

Abstract 1600: Molecular Characterization of PAX5 alterations in Adult BCR-ABL1-Positive Acute Lymphoblastic Leukemia (ALL)

Abstract Background: Recently, a genome-wide analysis using single nucleotide polymorphism (SNP) microarrays, identified genetic alterations targeting PAX5 in over 30% of pediatric acute lymphoblastic leukemia cases (Mullighan et al. Nature 2008). However, the occurrence of PAX5 alterations has not been investigated in adult BCR-ABL1-positive ALL. Aim: To characterize mutations and rearrangements on 9p involving PAX5 in adult BCR-ABL1-positive ALL. Patients and Methods: Eighty-nine patients with de novo adult BCR-ABL1-positive ALL were enrolled into institutional (n = 15) or Gruppo Italiano Malattie Ematologiche Maligne dell'Adulto Acute Leukemia Working Party (n = 74) clinical trials and after obtaining informed consent their genome was analyzed by SNP arrays (Affymetrix 250K NspI and SNP 6.0), FISH, genomic PCR and resequencing. Results: SNP array analysis identified a loss of PAX5 in 29 patients (33%). In all cases the deletion was heterozygous. Four patterns of PAX5 deletion were observed: focal deletion involving only the PAX5 gene in one case (3%); deletions involving only a portion of PAX5 and both telomeric and centromeric genes in 11 cases (38%) with a median size of 310 kb (range: 101 kb-16,395 kb); broader deletions involving the entire PAX5 locus and a variable number of flanking genes in 10 patients (34%) with a median size of 1,999 kb (range: 567 kb-1,208 kb); deletion of all chromosome 9 or 9p in 7 patients (24%). These deletions may result in PAX5 haploinsufficiency or altered gene expression. In one patient the deletion of PAX5 involved only a subset of exons (2-6) resulting in an alternative transcript encoding a prematurely truncated protein lacking key PAX5 functional domains. To investigate the consequences of genomic PAX5 alteration, quantitative PCR (q-PCR) was used: genomic alterations on 9p13.2 lead to a significant down-modulation at the transcript level of Pax5. Normal and deleted PAX5 subgroups were also investigated for point mutations, analyzing each exon by direct sequencing, but we did not found nucleotide substitutions. Conclusion: PAX5 deletions, but not point mutations, are frequent events in adult BCR-ABL1 positive ALL and are the main mechanism of inactivation of PAX5 in BCR-ABL1 positive ALL. Supported by: European LeukemiaNet, AIL, AIRC, FIRB 2006, Strategico di Ateneo, GIMEMA Onlus. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1600.

PAX5 Wild-Type without IKZF1 (Ikaros) Deletion Is Associated with Prolonged Disease-Free Survival and Low Rate of Cumulative Incidence of Relapse in Adult BCR-ABL1-Positive Acute Lymphoblastic Leukemia (ALL): On Behalf of GIMEMA AL Working Party.

Abstract 12 Background:Recently, in genome-wide analyses of DNA copy number abnormalities using single nucleotide polymorphism (SNP) microarrays, genetic alterations targeting PAX5 were identified in over 30% of pediatric acute lymphoblastic leukemia cases (Mullighan et al. Nature 2008). However, so far, the occurrence of PAX5 alterations and their clinical correlation has not been investigated in adults with BCR-ABL1-positive ALL. Aim:To characterize the rearrangements on 9p involving PAX5 and their clinical significance in adult BCR-ABL1-positive ALL. Patients and Methods:Eighty-nine patients with de novo adult BCR-ABL1-positive ALL were enrolled into institutional (n = 15) or Gruppo Italiano Malattie Ematologiche Maligne dell'Adulto Acute Leukemia Working Party (n = 74) clinical trials and after obtaining informed consent their genome was analyzed by SNP arrays (Affymetrix 250K NspI and SNP 6.0), FISH, genomic PCR and resequencing. Results:By SNP array analysis we identified a loss of PAX5 in 29 patients (33%). In all cases the deletion was hemizygous. Four patterns of PAX5 deletion were observed: 1) focal deletion involving only the PAX5 gene in one case (3%); 2) deletions involving only a portion of PAX5 and both telomeric and centromeric genes in 11 cases (38%) with a median size of 310 kb (range: 101 kb-16,395 kb); 3) broader deletions involving the entire PAX5 locus and a variable number of flanking genes in 10 patients (34%) with a median size of 1,999 kb (range: 567 kb-1,208 kb); 4) deletion of all chromosome 9 or 9p in 7 patients (24%). These deletions may result in either PAX5 haploinsufficiency or expression of PAX5 alleles with impaired DNA-binding or transcriptional activation activity. In one patient the deletion of PAX5 involved only a subset of exons (exons 2-6) resulting in an alternative transcript encoding a prematurely truncated protein lacking key PAX5 functional domains. To investigate the consequences of genomic PAX5 alteration, quantitative PCR (q-PCR) was used, demonstrating that genomic alterations on 9p13.2 lead to a significant down-modulation at the transcript level of Pax5. Furthermore, both normal and deleted PAX5 subgroups were investigated for point mutations by sequencing analysis but we did not found nucleotide substitutions, suggesting that deletions are the main mechanism of inactivation of PAX5 in BCR-ABL1 positive ALL. However, when we investigated the association of PAX5 deletions with clinical outcome, no association with PAX5 status was detected (overall survival: p=0.3294; disease free-survival,DFS: p=0.9249 and cumulative incidence of relapse, CIR: p=0.945), suggesting that PAX5 deletions are not associated with outcome. Twenty-three patients (26%) had deletion of both PAX5 and IKZF1, encoding for the transcription factor Ikaros. We assessed the contribution of both PAX5 and IKZF1 alterations on clinical outcome finding out that the occurrence of both normal PAX5 and IKZF1 loss is associated with a shorter DFS (p=0.04) and higher CIR cumulative incidence of relapse in comparison to patients with both normal IKZF1 and PAX5 (p = 0.009). These results were confirmed by multivariate analysis. Conclusion:PAX5 deletions are frequent events in adult BCR-ABL1 positive ALL and are often associated with IKZF1 loss. The genomic status of both PAX5 and IKZF1 can be useful to identify at diagnosis groups of patients with worse prognosis.Supported by: European LeukemiaNet, AIL, AIRC, FIRB 2006, Strategico di Ateneo, GIMEMA Onlus. Disclosures:No relevant conflicts of interest to declare.

P53 alterations in recurrent squamous cell cancer of the head and neck refractory to radiotherapy.

The aim of the study was to determine the incidence of p53 alterations by mutation, deletion or inactivation by mdm2 or human papillomavirus (HPV) infection in recurrent squamous cell cancer of the head and neck (SCCHN) refractory to radiotherapy. Twenty-two tumours were studied. The p53 status of each tumour was analysed by sequencing of exons 4–9 and by immunohistochemistry. Mdm2 expression was assessed by immunohistochemistry and HPV infection was assessed by polymerase chain reaction of tumour DNA for HPV 16, 18 and 33. Fifteen (68%) of the 22 tumours studied had p53 mutations, while seven had wild-type p53 sequence. p53 immunohistochemistry correlated with the type of mutation. HPV DNA was detected in 8 (36%) tumours and all were of serotype HPV 16. Of these, five were in tumours with mutant p53 and three were in tumours with wild-type p53. Mdm2 overexpression was detected in 11 (50%) tumours. Of these, seven were in tumours with mutant p53 and four were in tumours with wild-type p53. Overall, 21 of the 22 tumours had p53 alterations either by mutation, deletion or inactivation by mdm2 or HPV. In this study, the overall incidence of p53 inactivation in recurrent head and neck cancer was very high at 95%. The main mechanism of inactivation was gene mutation or deletion which occurred in 15 of the 22 tumours studied. In addition, six of the seven tumours with wild-type p53 sequence had either HPV 16 DNA, overexpression of mdm2 or both which suggested that these tumours had p53 inactivation by these mechanisms. This high incidence of p53 dysfunction is one factor which could account for the poor response of these tumours to radiotherapy and chemotherapy. Therefore, new therapies for recurrent SCCHN which either act in a p53 independent pathway, or which restore p53 function may be beneficial in this disease. © 2000 Cancer Research Campaign

Inactivation of p16/CDKN2 and p15/MTS2 genes in different histological types and clinical stages of primary ovarian tumors.

To define the involvement of p16/CDKN2 and p15/MTS2 inactivation in ovarian tumorigenesis and the association of these inactivation events with histological types and clinical stages of ovarian tumors, we analyzed homozygous deletion and somatic mutation of p16/CDKN2 and p15/MTS2 genes, as well as hypermethylation of the 5'-CpG island of the p16/CDKN2 gene, in 49 primary ovarian tumors and 6 ovarian carcinoma cell lines. We found homozygous deletions of p16/CDKN2 and p15/MTS2 in 6 (12%) and 5 (10%) primary tumors, respectively. Somatic mutation of p16/CDKN2 was found in only 1 primary tumor, but mutation of p15/MTS2 was not detected in any sample. None of the 28 primary tumors or 6 cell lines was hypermethylated at the 5'-CpG island of p16/CDKN2. The incidence of inactivation of p16/CDKN2 in primary tumors was significantly higher in the advanced stages (7 of 29) than in the early stages (0 of 14). Seven of 9 alterations in p16/CDKN2 and p15/MTS2 were observed in serous (3 of 12), endometrioid (3 of 9) and clear-cell (1 of 4) carcinomas. However, only normal sequences of these genes were detected in mucinous carcinomas. Loss of heterozygosity (LOH) at the IFNA locus was detected in 1 of 19 (5%) tumors, but no change at the D9S171 locus was observed in 17 tumors. These results suggest that: (i) homozygous deletion is the main mechanism of inactivation of p16/CDKN2 and p15/MTS2 in ovarian tumorigenesis; (ii) inactivation of p16/CDKN2 and p15/MTS2 may be the histological type-specific events involved in ovarian tumorigenesis; and (iii) inactivation of p16/CDKN2 is potentially involved in the progression of ovarian tumors in advanced stages.

Aconitase is readily inactivated by peroxynitrite, but not by its precursor, nitric oxide.

Mitochondrial and cytosolic aconitases have been indicated as major targets of .NO- and O2-.-mediated toxicity in cells due to the oxidant-mediated disruption of the [4Fe-4S] prosthetic group. However, under circumstances in which both .NO and O2-. are generated, their almost diffusion-controlled combination reaction (k = 6.7 x 10(9) M-1 s-1), leading to the formation of peroxynitrite anion (ONOO-), can out-compete the direct reactions of .NO and O2-. with aconitase and even the enzymatic dismutation of O2-. by superoxide dismutase. In this work, we report that ONOO- reacts with isolated pig heart mitochondrial aconitase at 1.4 x 10(5) M-1 s-1, resulting in a significant loss of enzymatic activity. Aconitase activity was totally recovered after postincubation with thiols and ferrous iron, indicating that ONOO- reactions with the enzyme involve the perturbation of the labile Fe alpha to yield the inactive [3Fe-4S] cluster, which is also evident by spectral changes. On the other hand, anaerobic exposure of isolated aconitase to high concentrations of .NO (> 100 microM) led to a moderate inhibition of the enzyme, which could be fully overcome by .NO displacement under an argon-saturated atmosphere, in agreement with the formation of a reversible inhibitory complex between .NO and the active site of aconitase. Superoxide inactivated mitochondrial aconitase at (3.5 +/- 2) x 10(6) M-1 s-1, a reaction rate 3 orders of magnitude slower than its reaction rate with .NO. O2-. could represent the main mechanism of inactivation of the enzyme in systems in which it is formed without significant concomitant production of .NO. Our results imply that the mechanisms by which .NO and O2-. inactivate aconitase in cell systems may not be simple due to their direct reactions with the iron-sulfur cluster, but may rely on the formation of ONOO-.

Fate and distribution of [ 14C] succinyl neocarzinostatin in rats

In vivo distribution of the [ 14 C] succinyl derivative of the antitumor antibiotic, neocar zinostatin (NCS), was studied in rats. The nature of the derivative is almost identical to the parental NCS. The radioactivity of the derivative within 20 min after i.v. injection was found to have accumulated in large amounts in such organs as the kidney, bladder, ovary—uterus, bone marrow, tongue, intestine, lung, skin, stomach and muscle. The levels in the plasma and bladder, although partly due to decomposed components, were high even after 1 hr. Thymus, spleen, and eye were found to contain only a small amount of this derivative. No appreciable amount of radioactivity was found in the brain. The accumulation of radioactivity in the kidney was greater than 6·9% of input. Within 20 min, 45·5% of the drug was found in urine, indicating a very rapid turnover. The desuccinylation of the derivative was much milder than anticipated while degradation by proteolysis of NCS was the main mechanism of inactivation which was followed by excretion. The nature of accumulated molecules in the kidney and bladder was largely high molecular weight components (mean weight > 6000). NCS seems to be excreted into the urine both in the degraded forms and in almost intact forms.