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...     

The alteration of PLCζ protein expression in unexplained infertile and asthenoteratozoospermic patients: A potential effect on sperm fertilization ability

Failed oocyte activation has been observed in unexplained infertile (UI) and asthenoteratozoospermic (AT) men. The deficiency of phospholipase C‐zeta (PLCζ) could be a possible reason for such failures and has not been studied yet. We investigated the expression and localization of PLCζ protein in the sperms of patients with UI and AT conditions. The relationships between PLCζ‐related parameters with male age, sperm characteristics, DNA integrity, and cellular maturity were assessed. Semen samples were collected from fertile (n = 40), UI (n = 40), and AT (n = 40) men. Subsequently, semen analysis, DNA fragmentation, hyaluronic acid‐binding ability, and PLCζ level along with its distribution were evaluated using computer‐assisted sperm analyzer, sperm chromatin structure assay (SCSA), hyaluronic acid‐binding assay (HBA), western blot analysis and immunofluorescence microscopy, respectively. Unlike SCSA, the values of HBA, and PLCζ expression were significantly reduced in UI and AT patients compared to fertile men, whereas no significant differences were observed among the experimental groups in terms of PLCζ localization patterns. The regression analysis also showed that HBA is the only variable associated with PLCζ levels. Furthermore, the correlation of male age with PLCζ localization in postacrosomal, equatorial, and acrosomal+postacrosomal+equatorial (A+PA+E) patterns, as well as the relation of normal morphology, with the (A+PA+E) pattern, remained in the regression model. Our findings indicated that reduced PLCζ level along with the increased DNA fragmentation and impaired maturation may be possible etiologies of decreased fertilization in the studied subjects.     

Serological tests for COVID-19: Potential opportunities

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel betacoronavirus, caused a pandemic leading to a standstill of nearly all global activities. There are some controversies on the production of specific immunoglobulin M (IgM) and IgG antibodies after the infection with SARS-CoV-2. This paper seeks to elaborate on the potential application of IgM and IgG antibodies and the viral antigens for the diagnosis and the course of the disease as well as the recurrence of positive nucleic acid tests after discharge.     

Generalized Baum-Welch Algorithm Based on the Similarity between Sequences

The profile hidden Markov model (PHMM) is widely used to assign the protein sequences to their respective families. A major limitation of a PHMM is the assumption that given states the observations (amino acids) are independent. To overcome this limitation, the dependency between amino acids in a multiple sequence alignment (MSA) which is the representative of a PHMM can be appended to the PHMM. Due to the fact that with a MSA, the sequences of amino acids are biologically related, the one-by-one dependency between two amino acids can be considered. In other words, based on the MSA, the dependency between an amino acid and its corresponding amino acid located above can be combined with the PHMM. For this purpose, the new emission probability matrix which considers the one-by-one dependencies between amino acids is constructed. The parameters of a PHMM are of two types; transition and emission probabilities which are usually estimated using an EM algorithm called the Baum-Welch algorithm. We have generalized the Baum-Welch algorithm using similarity emission matrix constructed by integrating the new emission probability matrix with the common emission probability matrix. Then, the performance of similarity emission is discussed by applying it to the top twenty protein families in the Pfam database. We show that using the similarity emission in the Baum-Welch algorithm significantly outperforms the common Baum-Welch algorithm in the task of assigning protein sequences to protein families.     

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.