Analysis of mitochondrial DNA from somatic hybrid cell lines of Saccharum officinarum (sugarcane) and Pennisetum americanum (pearl millet)

Mitochondrial DNA (mtDNA) from cell suspension cultures of two intergeneric somatic hybrids of Pennisetum americanum (pearl millet) + Saccharum officinarum (sugarcane) was examined by restriction endonuclease digestion and hybridization with sorghum mtDNA cosmids. The mtDNA of one somatic hybrid was indistinguishable from that of pearl millet, while the second exhibited a combination of parental mtDNAs, suggesting mitochondrial fusion. Several novel, possibly recombinant, mtDNA restriction fragments were detected in this hybrid, which may have resulted from intergenmic recombination.Florida Agriculture Experiment Station Journal Series No: 8090.     

Genetic transformation of a pasture legume,Lotus corniculatus,L. (birdsfoot trefoil)

Summary A rapid procedure for introducing foreign genes inLotus corniculatus based on the induction of hairy roots byAgrobacterium rhizogenes was developed. Expression of chloramphenicol acetyltransferase and neomycin phosphotransferase II was revealed in transgenic plants. Southern blot hybridization was used to confirm the genetic transformation. The transgenic plants looked normal and did not show any morphological modification compared to the seed grown plants.     

Transformation of the wild tomatoLycopersicon chilense Dun. byAgrobacterium tumefaciens

Summary Leaf disc transformation-regeneration technique was applied to the drought tolerant wild relative of cultivated tomato,Lycopersicon chilense, using a plasmid construct which contained the coding sequences of neomycin phosphotransferase (NPTII) and chloramphenicol acetyltransferase (CAT) genes. The two genotypes used, LA2747 and LA1930, showed a distinct difference in their aptitude to transformation; a higher success rate was obtained for the first genotype in every stage of the process. Shoots were formed on the regeneration medium containing 100 g/ml kanamycin through direct or indirect organogenesis. Root formation became only possible when the concentration of kanamycin was reduced to 50 g/ml. Expression of chloramphenicol acetyltransferase gene was observed in all of the kanamycin-screened plants after they matured; the activity of the gene was absent or low in some of the young plants. The presence of the CAT gene in transgenic plants was further confirmed by Southern blot analysis. Although transgenic plants grew to maturity, they did not produce fruit, owing to the self incompatibility ofL. chilense. Abbreviations BAP 6-benzylaminopurine - CAT chloramphenicol acetyltransferase - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - LB Luria Broth - EDTA ethylenediamine-tetraacetic acid     

The Human Cathelicidin LL-37, a Defensive Peptide Against Rotavirus Infection

International Journal of Peptide Research and Therapeutics - LL-37 is a 37 amino acid long cationic peptide belonging to the cathelicidin family of antimicrobial peptides. Limited investigations...     

Vibrio cholerae Evades Neutrophil Extracellular Traps by the Activity of Two Extracellular Nucleases

The Gram negative bacterium Vibrio cholerae is the causative agent of the secretory diarrheal disease cholera, which has traditionally been classified as a noninflammatory disease. However, several recent reports suggest that a V. cholerae infection induces an inflammatory response in the gastrointestinal tract indicated by recruitment of innate immune cells and increase of inflammatory cytokines. In this study, we describe a colonization defect of a double extracellular nuclease V. cholerae mutant in immunocompetent mice, which is not evident in neutropenic mice. Intrigued by this observation, we investigated the impact of neutrophils, as a central part of the innate immune system, on the pathogen V. cholerae in more detail. Our results demonstrate that V. cholerae induces formation of neutrophil extracellular traps (NETs) upon contact with neutrophils, while V. cholerae in return induces the two extracellular nucleases upon presence of NETs. We show that the V. cholerae wild type rapidly degrades the DNA component of the NETs by the combined activity of the two extracellular nucleases Dns and Xds. In contrast, NETs exhibit prolonged stability in presence of the double nuclease mutant. Finally, we demonstrate that Dns and Xds mediate evasion of V. cholerae from NETs and lower the susceptibility for extracellular killing in the presence of NETs. This report provides a first comprehensive characterization of the interplay between neutrophils and V. cholerae along with new evidence that the innate immune response impacts the colonization of V. cholerae in vivo. A limitation of this study is an inability for technical and physiological reasons to visualize intact NETs in the intestinal lumen of infected mice, but we can hypothesize that extracellular nuclease production by V. cholerae may enhance survival fitness of the pathogen through NET degradation.     

Crystal structure of native and a mutant of Lampyris turkestanicus luciferase implicate in bioluminescence color shift

Firefly bioluminescence reaction in the presence of Mg^2 +, ATP and molecular oxygen is carried out by luciferase. The luciferase structure alterations or modifications of assay conditions determine the bioluminescence color of firefly luciferase. Among different beetle luciferases, Phrixothrix hirtus railroad worm emits either yellow or red bioluminescence color. Sequence alignment analysis shows that the red-emitter luciferase from Phrixothrix hirtus has an additional arginine residue at 353 that is absent in other firefly luciferases. It was reported that insertion of Arg in an important flexible loop350–359 showed changes in bioluminescence color from green to red and the optimum temperature activity was also increased. To explain the color tuning mechanism of firefly luciferase, the structure of native and a mutant (E354R/356R/H431Y) of Lampyris turkestanicus luciferase is determined at 2.7 Å and 2.2 Å resolutions, respectively. The comparison of structure of both types of Lampyris turkestanicus luciferases reveals that the conformation of this flexible loop is significantly changed by addition of two Arg in this region. Moreover, its surface accessibility is affected considerably and some ionic bonds are made by addition of two positive charge residues. Furthermore, we noticed that the hydrogen bonding pattern of His431 with the flexible loop is changed by replacing this residue with Tyr at this position. Juxtaposition of a flexible loop (residues 351–359) in firefly luciferase and corresponding ionic and hydrogen bonds are essential for color emission.     

In Vitro and In Vivo Evaluation of a 18F-Labeled High Affinity NOTA Conjugated Bombesin Antagonist as a PET Ligand for GRPR-Targeted Tumor Imaging

Expression of the gastrin-releasing peptide receptor (GRPR) in prostate cancer suggests that this receptor can be used as a potential molecular target to visualize and treat these tumors. We have previously investigated an antagonist analog of bombesin (D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH₂, RM26) conjugated to 1,4,7-triazacyclononane-N,N'',N''''-triacetic acid (NOTA) via a diethylene glycol (PEG₂) spacer (NOTA-P2-RM26) labeled with ⁶⁸Ga and ¹¹¹In. We found that this conjugate has favorable properties for in vivo imaging of GRPR-expression. The focus of this study was to develop a ¹⁸F-labelled PET agent to visualize GRPR. NOTA-P2-RM26 was labeled with ¹⁸F using aluminum-fluoride chelation. Stability, in vitro binding specificity and cellular processing tests were performed. The inhibition efficiency (IC₅₀) of the [^natF]AlF-NOTA-P2-RM26 was compared to that of the ^natGa-loaded peptide using ¹²⁵I-Tyr⁴-BBN as the displacement radioligand. The pharmacokinetics and in vivo binding specificity of the compound were studied. NOTA-P2-RM26 was labeled with ¹⁸F within 1 h (60-65% decay corrected radiochemical yield, 55 GBq/µmol). The radiopeptide was stable in murine serum and showed high specific binding to PC-3 cells. [^natF]AlF-NOTA-P2-RM26 showed a low nanomolar inhibition efficiency (IC₅₀=4.4±0.8 nM). The internalization rate of the tracer was low. Less than 14% of the cell-bound radioactivity was internalized after 4 h. The biodistribution of [¹⁸F]AlF-NOTA-P2-RM26 demonstrated rapid blood clearance, low liver uptake and low kidney retention. The tumor uptake at 3 h p.i. was 5.5±0.7 %ID/g, and the tumor-to-blood, -muscle and -bone ratios were 87±42, 159±47, 38±16, respectively. The uptake in tumors, pancreas and other GRPR-expressing organs was significantly reduced when excess amount of non-labeled peptide was co-injected. The low uptake in bone suggests a high in vivo stability of the Al-F bond. High contrast PET image was obtained 3 h p.i. The initial biological results suggest that [¹⁸F]AlF-NOTA-P2-RM26 is a promising candidate for PET imaging of GRPR in vivo.     

Mn(II) complex of a vitamin B6 Schiff base as an exclusive apoptosis inducer in human MCF7 and HepG2 cancer cells: Synthesis,characterization, and biological studies

Herein, a Mn(II) complex of the N,N′-dipyridoxyl(1,4-butanediamine) (═H₂L) Schiff base has been newly synthesized. The synthesized complex was characterized by several experimental methods. In addition, the density functional theory approaches were used for theoretical identification of the complex. A good agreement between the computed and experimental infrared frequencies demonstrates validity of the optimized geometry for the synthesized complex. In a N₂O₂ manner, two azomethine nitrogens and two phenolate oxygens of the L²⁻ ligand are coordinated to the Mn²⁺ metal ion. The biological studies indicate an efficient apoptotic and antioxidant activities of the synthesized [MnL(CH₃OH)₂] complex on both of the HepG2 and MCF7 cancer cells. Since it has been suggested that the complex is an exclusive potent antitumor for treatment of the human breast and liver cancers.     

Colon cancer therapy by focusing on colon cancer stem cells and their tumor microenvironment

Despite many advances and optimization in colon cancer treatment, tumor recurrence and metastases make the development of new therapies necessary. Colon cancer stem cells (CCSCs) are considered as the main triggering factor of cancer progression, recurrence, and metastasis. CCSCs as a result of accumulated genetic and epigenetic alterations and also complex interconnection with the tumor microenvironment (TME) can evolve and convert to full malignant cells. Mounting evidence suggests that in cancer therapy both CCSCs and non-CCSCs in TME have to be regarded to break through the limitation of current therapies. In this regard, stem cell capabilities of some non-CCSCs may arise inside the TME condition. Therefore, a deep knowledge of regulatory mechanisms, heterogeneity, specific markers, and signaling pathways of CCSCs and their interconnection with TME components is needed to improve the treatment of colorectal cancer and the patient's life quality. In this review, we address current different targeted therapeutic options that target cell surface markers and signaling pathways of CCSCs and other components of TME. Current challenges and future perspectives of colon cancer personalized therapy are also provided here. Taken together, based on the deep understanding of biology of CCSCs and using three-dimensional culture technologies, it can be possible to reach successful colon cancer eradication and improvise combination targeted therapies against CCSCs and TME.