The differential catalytic activity of ribosome-inactivating proteins saporin 5 and 6 is due to a single substitution at position 162

Saporin, a type I ribosome-inactivating protein produced by the soapwort plant Saponaria officinalis belongs to a multigene family that encodes its several isoforms. The saporin seed isoform 6 has significantly higher N-glycosidase and cytotoxic activities compared with the seed isoform 5, although the two have identical active sites. In the present study, we have investigated the contribution of non-conservative amino acid changes outside the active sites of these isoforms towards their differential catalytic activity. The saporin 6 residues Lys134, Leu147, Phe149, Asn162, Thr188 and Asp196 were replaced by the corresponding saporin 5 residues, Gln134, Ser147, Ser149, Asp162, Ile188 and Asn196, to generate six variants of saporin 6, K134Q, L147S, F149S, N162D, T188I and D196N. By functional characterization, we show that the change in amino acid Asn162 in saporin 6 to aspartic acid residue of saporin 5 contributes mainly to the lower catalytic activity of saporin 5 compared with saporin 6. The non-involvement of other non-conservative amino acids in the differential catalytic activity of these isoforms was confirmed with the help of the double mutations N162D/K134Q, N162D/L147S, N162D/F149S, N162D/T188I and N162D/D196N

Interaction of human pancreatic ribonuclease with human ribonuclease inhibitor

Mammalian ribonucleases interact very strongly with the intracellular ribonuclease inhibitor (RI). Eukaryotic cells exposed to mammalian ribonucleases are protected from their cytotoxic action by the intracellular inhibition of ribonucleases by RI. Human pancreatic ribonuclease (HPR) is structurally and functionally very similar to bovine RNase A and interacts with human RI with a high affinity. In the current study, we have investigated the involvement of Lys-7, Gln-11, Asn-71, Asn-88, Gly-89, Ser-90, and Glu-111 in HPR in its interaction with human ribonuclease inhibitor. These contact residues were mutated either individually or in combination to generate mutants K7A, Q11A, N71A, E111A, N88R, G89R, S90R, K7A/E111A, Q11A/E111A, N71A/E111A, K7A/N71A/E111A, Q11A/N71A/E111A, and K7A/Q11A/N71A/E111A. Out of these, eight mutants, K7A, Q11A, N71A, S90R, E111A, Q11A/E111A, N71A/E111A, and K7A/N71A/E111A, showed an ability to evade RI more than the wild type HPR, with the triple mutant K7A/N71A/E111A having the maximum RI resistance. As a result, these variants exhibited higher cytotoxic activity than wild type HPR. The mutation of Gly-89 in HPR produced no change in the sensitivity of HPR for RI, whereas it has been reported that mutating the equivalent residue Gly-88 in RNase A yielded a variant with increased RI resistance and cytotoxicity. Hence, despite its considerable homology with RNase A, HPR shows differences in its interaction with RI. We demonstrate that interaction between human pancreatic ribonuclease and RI can be disrupted by mutating residues that are involved in HPR-RI binding. The inhibitor-resistant cytotoxic HPR mutants should be useful in developing therapeutic molecules

Protein Kinase C Epsilon Overexpression in Prostate Adenocarcinoma is Associated with Oncogenesis

Background: PKCε, an isozyme of serine-threonine kinase, has been implicated in epithelial cancer metastasis and progression. This study investigates the impact of the oncogenic PKCε, overexpressed abnormally in human Prostate tumor samples and cell lines, to understand its efficacy. Methods: The microarray dataset, GSE86257, was processed for normalization. The identification of upregulated and downregulated genes was based on FDR >1 and p <0.05 values. Cytoscape analysis and functional enrichment of significant genes were done. The identified genes were validated on the TCGA dataset and survival analysis was performed by Kaplan-Meier analysis. Results: A total of 1524 DEGs were identified with 728 upregulated genes and 818 downregulated genes. The two significant modules with MCODE score:9.0 and Venn analysis provided cyclin-dependent kinase inhibitor protein (CDK1), Cyclin B1 (CCNB1), Phospholipase C Gamma 1 (PLCG1), Cyclin Dependent Kinase 9 (CDK9), Phosphoinositide-3-Kinase Regulatory Subunit 3 (PIK3R3), H4 Clustered Histone 6 (H4C6), Phospholipase C Gamma 2 (PLCG2) as most interacting genes. TCGA data analysis and Prognostic analysis revealed CCNBI, CDK9, and PLCG1 associated with poor prognosis. Conclusion: PKCε regulates genes that are responsible for cancer progression. Therefore, targeting PKCε in Prostate cancer may serve as an important regulatory effect and may improve the prognosis of the disease.&nbsp

Growth-promoting activity of desmopressin in murine leukemia cells treated in vitro

The synthetic vasopressin analogue, desmopressin (dDAVP), has been shown to influence membrane transport of melphalan in murine L5178Y lymphoblasts. Accordingly, the effect of dDAVP on the cytocidal activity of melphalan in L5178Y cells was evaluated. dDAVP did not affect the cytocidal activity of melphalan in these cells, but significantly affected the cloning efficiency of stationary phase or slowly dividing L5178Y cells over a range of concentrations. In particular, stationary phase cells showed an increase in cloning efficiency from 4.3 ± 0.5% in control cells to 7.0 ± 0.3% in cells treated with 25 nM dDAVP (P < 0.001), whereas cells doubling every 26 h showed an increase from 10.8 ± 1.2% in control cells to 21.0 ± 2.0% in cells treated with 150 nM dDAVP (P < 0.001). This phenomenon was associated with significant elevations of 1,2[3H] diacylglycerol after incubation with dDAVP for 9 min (P < 0.01) and total [3H]diacylglycerols after incubation for both 3 min (P < 0.05) and 9 min (P < 0.02). Within 10 s of treatment with 100 nM dDAVP, there was a marked decrease in the levels of inositol 1,4,5-trisphosphate and inositol 1-phosphate, but subsequently no change was observed for up to 9 min after treatment. We postulate that the increase of diacylglycerol content produced by dDAVP might be primarily from a phosphatidylcholine source and that the growth-promoting activity of desmopressin may be a consequence of activation of protein kinase C

Antitumor effects of B3-PE and B3-lysPE40 in a nude mouse model of human breast cancer and the evaluation of B3-PE toxicity in monkeys

B3 is a tumor-reactive monoclonal antibody (mAb) that binds to a limited number of normal tissues.Immunotoxins made with B3 coupled to either Pseudomonas exotoxin (PE) or recombinant forms of PE with a deletion of the cell-binding domain (LysPE40) have been shown to cause complete tumor regression in nude mice bearing a rapidly growing A431 (L. H. Pai et al., Proc. Natl. Acad. Sci. USA, 88: 3358-3362, 1991) human epidermoid carcinoma. In this study we show that an immunotoxin composed of mAb B3 when chemically coupled to LysPE40 (B3-LysPE40) led to complete regression of a slowly growing breast cancer, MCF-7, in nude mice when given i.v. every other day for five doses. mAb B3 coupled to native PE also produced significant regression of the MCF-7 tumor. The reactivity of mAb B3 was evaluated using an immunohistochemical method on the two responsive tumors, MCF-7 and A431, and compared with a typical human colon carcinoma specimen that has B3 antigen on its surface. The results showed that both A431 and MCF-7 xenograft tumors have similar reactivity to B3 when compared with the human colon carcinoma specimen. To evaluate the toxicity of B3-PE in primates, Cynomolgus monkeys received escalating doses of B3-PE i.v. on Days 1, 3, and 5. Based on antibody localization studies using frozen sections of normal human and monkey tissue, gastric, trachea, and bladder mucosal injury could have occurred. However, no clinical signs of injury or histological damage to these organs were seen at the doses administered. Chemical hepatitis due to PE was transient and well tolerated at doses up to 50 μg/kg for three doses. The lethal dose was about 100 μg/kg, and the cause of death was liver necrosis, as shown by necropsy. We conclude that m Ab B3, when coupled to PE40 or PE, can produce strong antitumor activity in vivo. The similar level of reactivity of the B3 antibody in our tumor models with a surgical specimen of a human colon carcinoma and the toxicity study in monkeys indicate that therapeutic doses of B3-PE and B3-LysPE40 can be delivered without causing toxicity to normal organs that express B3 antigen. Although both B3-PE and B3-LysPE40 have antitumor activity in nude mice bearing a human xenograft, B3-LysPE40 is better tolerated and should be further evaluated as a therapeutic agent for cancer patients

Multifactorial resistance to adriamycin: relationship of DNA repair, glutathione transferase activity, drug efflux, and p-glycoprotein in cloned cell lines of adriamycin-sensitive and -resistant P388 Leukemia

Cloned lines of Adriamycin (ADR)-sensitive and -resistant P388 leukemia have been established, including P388/ADR/3 and P388/ADR/7 that are 5- and 10-fold more resistant than the cloned sensitive cell line P388/4 (Cancer Res., 46: 2978, 1986). A time course of ADR-induced DNA double-strand breaks revealed that in sensitive P388/4 cells, evidence of DNA repair was noted 4 h after removal of drug, whereas in resistant clone 3 and 7 cells repair was observed 1 h after drug removal. The earlier onset of DNA repair was statistically significant (p = 0.0154 for clone 3 cells, and p = 0.0009 for clone 7 cells). By contrast, once the repair process was initiated, the rate of repair was similar for all three cell lines. The level of glutathione transferase activity was determined in whole cell extracts. Enzyme activity (mean ± SE) in sensitive cells was 9.49 ± 1.00 nmol/min/mg protein, that in resistant clone 3 cells was 13.36 ± 1.03 nmol/min/mg, and that in clone 7 cells was 13.96 ± 1.44 nmol/min/mg; the 1.44-fold increase in enzyme activity in resistant cells was statistically significant (p = 0.01). Further evidence of induction of glutathione transferase was provided by Northern blot analysis using a 32P-labeled cDNA for an anionic glutathione transferase, which demonstrated approximately a twofold increase in mRNA in resistant clone 7 cells. Western blot analysis with a polyvalent antibody against anionic glutathione transferase also revealed a proportionate increase in gene product in resistant cells. Dose-survival studies showed that ADR-resistant cells were cross-resistant to actinomycin D, daunorubicin, mitoxantrone, colchicine, and etoposide, but not to the alkylating agent melphalan; this finding provided evidence that these cells are multidrug resistant. Using a cDNA probe for P-glycoprotein, a phenotypic marker for multidrug resistance, Northern blot analysis showed an increase in the steady state level of mRNA of approximately twofold in resistant clone 3 and 7 cells. Southern analysis with the same cDNA probe showed no evidence of gene amplification or rearrangement. Western blot analysis with monoclonal C219 antibody demonstrated a distinct increase in P-glycoprotein in resistant cells. Efflux of Adriamycin as measured by the efflux rate constant was identical in all three cell lines. Furthermore, the metabolic inhibitors azide and dinitrophenol did not augment drug uptake in either sensitive or resistant cells. These findings suggest that despite the increase in P-glycoprotein, an active extrusion pump was not operational in these cells. This and previous studies provide unequivocal evidence that resistance to Adriamycin is multifactorial. Decreased drug uptake, decreased formation of DNA single- and double-strand breaks, increased glutathione transferase activity, earlier onset of DNA repair, as well as elevated P-glycoprotein are all characteristic of multifactorial drug resistance

The C-terminus of ClpC1 of Mycobacterium tuberculosis is crucial for its oligomerization and function.

Mycobacterium tuberculosis ClpC1 is a member of the Hsp100/Clp AAA+ family of ATPases. The primary sequence of ClpC1 contains two N-terminal domains and two nucleotide binding domains (NBD). The second NBD has a long C-terminal sub-domain containing several motifs important for substrate interaction. Generally, ClpC proteins are highly conserved, however presence of C-terminal domains of variable lengths is a remarkable difference in ClpC from different species. In this study, we constructed deletion mutants at the C-terminus of M. tuberculosis ClpC1 to determine its role in the structure and function of the protein. In addition, a deletion mutant having the two conserved N-terminal domains deleted was also constructed to investigate the role of these domains in M. tuberculosis ClpC1 function. The N-terminal domains were found to be dispensable for the formation of oligomeric structure, and ATPase and chaperone activities. However, deletions beyond a specific region in the C-terminus of the ClpC1 resulted in oligomerization defects and loss of chaperonic activity of the protein without affecting its ATPase activity. The truncated mutants, defective in oligomerization were also found to have lost the chaperonic activity, showing the formation of oligomer to be required for the chaperonic activity of M. tuberculosis ClpC1. The current study has identified a region in the C-terminus of M. tuberculosis ClpC1 which is essential for its oligomerization and in turn its function