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1.
The current treatment strategies, chemotherapy and radiation therapy being used for the management of cancer are deficient
in targeted approach leading to treatment related toxicities and relapse. Contrarily, fusion toxins exhibit remarkable tumor
specificity thus emerging as an alternative therapy for the treatment of cancer. Diphtheria toxin-HN-1 peptide (DT/HN-1) is
a fusion toxin designed to target the head and neck squamous cell carcinoma (HNSCC). The aim of this study was to construct,
characterize, and evaluate the cytotoxicity and specificity of DT/HN-1 fusion toxin against the HNSCC cells. The purified
DT/HN-1 fusion toxin was characterized by SDS-PAGE and western blotting. Refolding of purified fusion toxins was monitored
by fluorescence spectra and circular dichroism spectra. The activity of DT/HN-1 fusion toxin was demonstrated on various HNSCC
cell lines by cell viability assay, cell proliferation assay, protein synthesis inhibition assay, apoptosis and cell cycle
analysis. The fusion toxin DT/HN-1 demonstrated remarkably high degree of cytotoxicity specific to the HNSCC cells. The IC50 of DT/HN-1 fusion toxin was ~1 to 5 nM in all the three HNSCC cell lines. The percentage apoptotic cells in DT/HN-1 treated
UMB-SCC-745 cells are 16% compared to 4% in untreated. To further demonstrate the specific toxicity of DT/HN-1 fusion toxin
towards the HNSCC cells we constructed, characterized and evaluated the efficacy of DT protein. The DT protein coding for
only a fragment of diphtheria toxin without its native receptor binding domain failed to exhibit any cytotoxicity on all the
cell lines used in this study thus establishing the importance of a ligand in achieving targeted toxicity. To evaluate the
translocation ability of HN-1 peptide, an additional construct DTΔT/HN-1 was constructed, characterized and evaluated for
its cytotoxic activity. The fusion toxin DTΔT/HN-1 deficient of the translocation domain of diphtheria toxin showed no cytotoxicity
on all the cell lines clearly indicating the inability of HN-1 peptide to translocate catalytic domain of the toxin into the
cytosol. 相似文献
2.
Pradeep Koppolu Sunkara Sirisha Ashank Mishra Kopparesh Deshpande Amara Swapna Lingam Dalal H. Alotaibi Mohammed Saleh Alwahibi Soumya Penela 《Saudi Journal of Biological Sciences》2021,28(1):380-385
Periodontitis is commonly diagnosed based on clinical parameters. However, the analysis of a few unique biomarkers of the disease process present in the saliva and blood can further assist the estimation of the rate of disease progression.AimThe present study attempted to correlate the alkaline phosphatase (ALP) and acid phosphatase (ACP) levels in saliva and serum between patients with healthy periodontium, gingivitis, and chronic periodontitis.Materials and methodsThe present study was conducted in 135 subjects between 20 and 55 years of age. The subjects were divided into three groups, namely healthy (Group A), gingivitis (Group B), and chronic periodontitis (Group C). The clinical parameters were recorded using the plaque index (PI), gingival index (GI), and probing depth (PD). Saliva and serum were analyzed for ALP and ACP levels using an auto analyzer. All patients underwent scaling and root planning (SRP) along with oral hygiene instructions. Patients were then recalled after four weeks, and blood and saliva samples were collected to estimate ALP and ACP levels prior to clinical examination.ResultsThe clinical parameters exhibited a statistically significant decrease in the PI and GI in both group B and group C after SRP. A significant change in the PD and attachment levels (AL) was observed in the periodontitis group after SRP. The mean salivary & serum ALP levels exhibited a statistically significant decrease in group B & C after SRP. The mean serum ACP levels exhibited a statistically significant decrease in group B & C after SRP However, the salivary ACP levels decrease after SRP was only statistically significant in group C.ConclusionSerum and salivary ALP and ACP levels were markedly decreased in the gingivitis and periodontitis groups after SRP and were positively correlated with the clinical parameters. 相似文献
3.
Lucy A. Jury Brett Sitzlar Sirisha Kundrapu Jennifer L. Cadnum Kim M. Summers Christine P. Muganda Abhishek Deshpande Ajay K. Sethi Curtis J. Donskey 《PloS one》2013,8(7)
Background
Recent reports suggest that community-associated Clostridium difficile infection (CDI) (i.e., no healthcare facility admission within 90 days) may be increasing in frequency. We hypothesized that outpatient clinics could be an important source for acquisition of community-associated CDI.Methods
We performed a 6-month prospective study of CDI patients to determine frequency of and risk factors for skin and environmental shedding during outpatient visits and to derive a prediction rule for positive cultures. We performed a point–prevalence culture survey to assess the frequency of C. difficile contamination in outpatient settings and evaluated the frequency of prior outpatient visits in patients with community-associated CDI.Results
Of 67 CDI patients studied, 54 (81%) had 1 or more outpatient visits within 12 weeks after diagnosis. Of 44 patients cultured during outpatient visits, 14 (32%) had skin contamination and 12 (27%) contaminated environmental surfaces. Decreased mobility, fecal incontinence, and treatment with non-CDI antibiotics were associated with positive cultures, whereas vancomycin taper therapy was protective. In patients not on CDI therapy, a prediction rule including incontinence or decreased mobility was 90% sensitive and 79% specific for detection of spore shedding. Of 84 clinic and emergency department rooms cultured, 12 (14%) had 1 or more contaminated environmental sites. For 33 community-associated CDI cases, 31 (94%) had an outpatient visit during the 12 weeks prior to onset of diarrhea.Conclusions
Patients with recent CDI present a significant risk for transmission of spores during outpatient visits. The outpatient setting may be an underappreciated source of community-associated CDI cases. 相似文献4.
Abdel-Wahab Z Kalady MF Emani S Onaitis MW Abdel-Wahab OI Cisco R Wheless L Cheng TY Tyler DS Pruitt SK 《Cellular immunology》2003,224(2):86-97
Modification of the parental immunodominant Melan-A/MART-1 peptide (MART-1(26-35)) by replacing the alanine with leucine (A27L) enhances its immunogenicity. Because of the reported advantages of RNA over peptides in DC vaccines, we sought to mutate the MART-1 gene to encode a full-length MART-1 antigen with an A27L amino acid substitution. Human DC were transfected with A27L-mutated MART-1 RNA (A27L RNA) or native MART-1 RNA, and then used to stimulate autologous T cells from a series of 8 HLA-A2+ volunteers. After three stimulations, all CTL induced with DC/A27L RNA exhibited more tetramer+ cells, and demonstrated stronger antigen-specific IFNgamma-secreting activity compared to CTL induced with DC/native RNA. A potent MART-1-specific, and predominantly class-I-restricted lysis was detected in most CTL induced with DC/A27L RNA, while native RNA-induced CTL showed minimal and non-specific lysis. HLA-A2+ DC and MART-1 negative/A2+ melanoma cells transfected with the A27L RNA were recognized and killed by MART-1-specific CTL, suggesting that these APC efficiently processed the A27L RNA and presented correct MART-1-specific epitope(s). In summary, introducing an A27L mutation into the MART-1 full-length mRNA sequence enhanced the immunogenicity of the encoded MART-1 Ag. The ease with which such a mutation can be made in RNA presents another potential advantage of using RNA for immunotherapy. Our results support considering this strategy for enhancing the immunogenicity of DC-based RNA vaccines. 相似文献
5.
Harsha L. Rao Viquar U. Begum Deepa Khadka Anil K. Mandal Sirisha Senthil Chandra S. Garudadri 《PloS one》2015,10(5)
PurposeTo compare the rate of mean deviation (MD) change on 24-2 versus 10-2 VFs in treated glaucomatous eyes with 5 or more examinations.MethodsIn a retrospective study, 24-2 and 10-2 VFs of 131 glaucoma patients (167 eyes) who had undergone at least 5 VFs examinations during their follow-up were analyzed. All these patients had VF defects both on 24-2 and 10-2 VFs. Rates of MD change were calculated using best linear unbiased predictions (BLUP).ResultsMedian age, MD on 24-2 VF at baseline, number of VFs performed during follow-up and follow-up duration were 55 years, -16.9 dB, 9 and 9 years respectively. Median rate of MD change was significantly greater (p<0.001) on 10-2 VF (-0.26 dB/year; interquartile range [IQR]: -0.47, -0.11) compared to 24-2 VFs (-0.19 dB/year; IQR: -0.41, -0.03). Comparing the rates of MD change in eyes with different severities of VF loss (early [MD better than -6 dB], moderate [-6 dB to -12 dB], advanced [-12 to -20 dB] and severe [MD worse than -20 dB]) at baseline (based on the MD on 24-2 VF), median rate of MD change was comparable between 10-2 and 24-2 VFs in mild (-0.45 dB/year vs. -0.40 dB/year, P = 0.42) and moderate (-0.32 dB/year vs. -0.40 dB/year, P = 0.26) VF loss categories, while the same were significantly greater on 10-2 VFs in advanced (-0.28 dB/year vs. -0.21 dB/year, P = 0.04) and severe (-0.18 dB/year vs. -0.06 dB/year, P<0.001) VF loss categories.ConclusionsIn patients with VF defects both on 24-2 and 10-2 VFs, evaluating the rate of MD change on 10-2 VFs may help in better estimation of glaucoma progression, especially so in eyes with advanced glaucoma at baseline. 相似文献
6.
A bioinformatic filter for improved base-call accuracy and polymorphism detection using the Affymetrix GeneChip® whole-genome resequencing platform
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Gagan A. Pandya Michael H. Holmes Sirisha Sunkara Andrew Sparks Yun Bai Kathleen Verratti Kelly Saeed Pratap Venepally Behnam Jarrahi Robert D. Fleischmann Scott N. Peterson 《Nucleic acids research》2007,35(21):e148
DNA resequencing arrays enable rapid acquisition of high-quality sequence data. This technology represents a promising platform for rapid high-resolution genotyping of microorganisms. Traditional array-based resequencing methods have relied on the use of specific PCR-amplified fragments from the query samples as hybridization targets. While this specificity in the target DNA population reduces the potential for artifacts caused by cross-hybridization, the subsampling of the query genome limits the sequence coverage that can be obtained and therefore reduces the technique's resolution as a genotyping method. We have developed and validated an Affymetrix Inc. GeneChip® array-based, whole-genome resequencing platform for Francisella tularensis, the causative agent of tularemia. A set of bioinformatic filters that targeted systematic base-calling errors caused by cross-hybridization between the whole-genome sample and the array probes and by deletions in the sample DNA relative to the chip reference sequence were developed. Our approach eliminated 91% of the false-positive single-nucleotide polymorphism calls identified in the SCHU S4 query sample, at the cost of 10.7% of the true positives, yielding a total base-calling accuracy of 99.992%. 相似文献
7.
Yeast aconitase in two locations and two metabolic pathways: seeing small amounts is believing
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The distribution of identical enzymatic activities between different subcellular compartments is a fundamental process of living cells. At present, the Saccharomyces cerevisiae aconitase enzyme has been detected only in mitochondria, where it functions in the tricarboxylic acid (TCA) cycle and is considered a mitochondrial matrix marker. We developed two strategies for physical and functional detection of aconitase in the yeast cytosol: 1) we fused the alpha peptide of the beta-galactosidase enzyme to aconitase and observed alpha complementation in the cytosol; and 2) we created an ACO1-URA3 hybrid gene, which allowed isolation of strains in which the hybrid protein is exclusively targeted to mitochondria. These strains display a specific phenotype consistent with glyoxylate shunt elimination. Together, our data indicate that yeast aconitase isoenzymes distribute between two distinct subcellular compartments and participate in two separate metabolic pathways; the glyoxylate shunt in the cytosol and the TCA cycle in mitochondria. We maintain that such dual distribution phenomena have a wider occurrence than recorded currently, the reason being that in certain cases there is a small fraction of one of the isoenzymes, in one of the locations, making its detection very difficult. We term this phenomenon of highly uneven isoenzyme distribution "eclipsed distribution." 相似文献
8.
Prokaryotes and lower eukaryotes possess redundant activities that remove the plethora of oxidative DNA base damages produced during normal oxidative metabolism and which have been associated with cancer and aging. Thus far, only one oxidized pyrimidine-specific DNA glycosylase has been identified in humans, hNthl. Here, we report the identification of three new putative human DNA glycosylases that are phylogenetically members of the Fpg/Nei family primarily found in the bacterial kingdom. We have characterized one of these, hNEI1, and show it to be functionally homologous to bacterial Nei, that is, its principal substrates are oxidized pyrimidines, it undergoes a lyase reaction by, beta,delta-elimination and traps a Schiff base with a substrate containing thymine glycol (Tg). Furthermore, inactivation of active site residues shown to be important in Escherichia coli Nei inactivate the human enzyme. The hNEI1 gene is located on the long arm of chromosome 15 that is frequently deleted in human cancers. 相似文献
9.
S. Prashant M. Sri Lakshmi Sunita V. L. Sirisha V. Vijaya Bhaskar A. Maruthi Rao M. Lakshmi Narasu P. B. Kavi Kishor 《Plant Cell, Tissue and Organ Culture》2012,108(3):421-436
Promoter sequences of a 795 bp cinnamoyl CoA reductase (LlCCR) and 1,882 bp cinnamyl alcohol dehydrogenase (LlCAD) genes were isolated from Leucaena leucocephala, a leguminous tree species by genome walking, and analysed using bioinformatics tools. This revealed presence of cis-elements such as AC-boxes, XYLAT, WRKY, and MYB binding sites in addition to CAAT and TATA boxes. For functional characterization,
each of LlCCR and LlCAD promoter sequences were fused to β-glucuronidase (GUS) reporter gene, immobilized into pBI101 plasmid, and introduced into tobacco via Agrobacterium tumefaciens strain LBA4404. Histochemical observations of transgenic lines indicated tissue-specific expression of GUS in the vascular
tissues of leaves, stems, and roots. These results demonstrate that GUS expression driven by either LlCCR or LlCAD promoters were involved in lignifying tissues, and more specifically in differentiating xylem cells. This observed tissue-specific
expression driven by either LlCCR or LlCAD promoters is sufficient for reducing the lignin content only in vascular tissues, thus overcoming the risks and challenges
associated with down-regulation of lignin content in whole plants. 相似文献
10.
Nidhi Batra Manuel A. Riquelme Sirisha Burra Rekha Kar Sumin Gu Jean X. Jiang 《The Journal of biological chemistry》2014,289(15):10582-10591
Connexin (Cx) 43 hemichannels in osteocytes are thought to play a critical role in releasing bone modulators in response to mechanical loading, a process important for bone formation and remodeling. However, the underlying mechanism that regulates the opening of mechanosensitive hemichannels is largely unknown. We have recently shown that Cx43 and integrin α5 interact directly with each other, and activation of PI3K appears to be required for Cx43 hemichannel opening by mechanical stimulation. Here, we show that mechanical loading through fluid flow shear stress (FFSS) increased the level of active AKT, a downstream effector of PI3K, which is correlated with the opening of hemichannels. Both Cx43 and integrin α5 are directly phosphorylated by AKT. Inhibition of AKT activation significantly reduced FFSS-induced opening of hemichannels and disrupted the interaction between Cx43 and integrin α5. Moreover, AKT phosphorylation on Cx43 and integrin α5 enhanced their interaction. In contrast to the C terminus of wild-type Cx43, overexpression of the C-terminal mutant containing S373A, a consensus site previously shown to be phosphorylated by AKT, failed to bind with α5 and hence could not inhibit hemichannel opening. Together, our results suggest that AKT activated by FFSS directly phosphorylates Cx43 and integrin α5, and Ser-373 of Cx43 plays a predominant role in mediating the interaction between these two proteins and Cx43 hemichannel opening, a crucial step to mediate the anabolic function of mechanical loading in the bone. 相似文献