Short and Straightforward Synthesis of Triphosphates of Artificial Nucleobase Pairs Displaying Unconventional Pairing Scheme

Concise, facile and efficient synthesis of 5′-O-triphosphates of 6-amino-5-nitro-3-(1′-β-D-2′-deoxyribofuranosyl)-2(1H)-pyridone (dZ) and its Watson-Crick complement 2-amino-8-(1′-β-D-2′-deoxyribofuranosyl)-imidazo[1,2a]-1,3,5-triazin-4(8H)-one (dP) is reported using a one-pot synthetic procedure.     

Distinct glycosyltransferases synthesize E-selectin ligands in human vs. mouse leukocytes

The binding of selectins to carbohydrate epitopes expressed on leukocytes is the first step in a multi-step cell adhesion cascade that controls the rate of leukocyte recruitment at sites of inflammation. The glycans that function as selectin-ligands are post-translationally synthesized by the serial action of Golgi resident enzymes called glycosyltransferases (glycoTs). Whereas much of our current knowledge regarding the role of glycoTs in constructing selectin-ligands comes from reconstituted biochemical investigations or murine models, tools to assess the impact of these enzymes on the human ligands are relatively underdeveloped. This is significant since the selectin-ligands, particularly those that bind E-selectin, vary between different leukocyte cell populations and they are also different in humans compared with mice. To address this shortcoming, a recent study by Buffone et al. (2013) outlines a systematic strategy to knockdown upto three glycoTs simultaneously in human leukocytes. The results suggest that the fucosyltransferases (FUTs) regulating selectin-ligand synthesis may be species-specific. In particular, they demonstrate that FUT9 plays a significant role during human, but not mouse, leukocyte-endothelial interactions. Overall, this article discusses the relative roles of the FUTs during human L-, E-, and P-selectin-ligand biosynthesis, and the potential that the knockdown strategy outlined here may assess the role of other glycoTs in human leukocytes also.     

A2B adenosine receptor blockade inhibits growth of prostate cancer cells

The role of the A_2B adenosine receptor (AR) in prostate cell death and growth was studied. The A_2B AR gene expression quantified by real-time quantitative RT-PCR and Western blot analysis was the highest among four AR subtypes (A₁, A_2A, A_2B, and A₃) in all three commonly used prostate cancer cell lines, PC-3, DU145, and LNCaP. We explored the function of the A_2B AR using PC-3 cells as a model. The A_2B AR was visualized in PC-3 cells by laser confocal microscopy. The nonselective A_2B AR agonist NECA and the selective A_2B AR agonist BAY60-6583, but not the A_2A AR agonist CGS21680, concentration-dependently induced adenosine 3′,5′-cyclic monophosphate (cyclic AMP) accumulation. NECA diminished lactate dehydrogenase (LDH) release, TNF-α-induced increase of caspase-3 activity, and cycloheximide (CHX)-induced morphological changes typical of apoptosis in PC-3 cells, which were blocked by a selective A_2B AR antagonist PSB603. NECA-induced proliferation of PC-3 cells was diminished by siRNA specific for the A_2B AR. The selective A_2B AR antagonist PSB603 was shown to inhibit cell growth in all three cell lines. Thus, A_2B AR blockade inhibits growth of prostate cancer cells, suggesting selective A_2B AR antagonists as potential novel therapeutics.     

Partial Purification and Characterization of a Bacteriocin DT24 Produced by Probiotic Vaginal Lactobacillus brevis DT24 and Determination of its Anti-Uropathogenic Escherichia coli Potential

The emergence of antibiotic resistance has increased the interest for finding new antimicrobials in the past decade. Probiotic Lactic acid bacteria producing antimicrobial proteins like bacteriocin can be excellent agents for development as novel therapeutic agents and complement to conventional antibiotic therapy. Uropathogenic Escherichia coli, most causative agent of Urinary tract infection, has developed resistance to various antibiotics. In the present investigation, antibacterial substance like bacteriocin (Bacteriocin DT24) produced by probiotic Lactobacillus brevis DT24 from vaginal sample of healthy Indian woman was partially purified and characterized. It was efficiently working against various pathogens, that is, Uropathogenic E. coli, Enterococcus faecium, Enterococcus faecalis and Staphylococcus aureus. The antimicrobial peptide was relatively heat resistant and also active over a broad range of pH 2–10. It has been partially purified by ammonium sulfate precipitation and gel filtration chromatography and checked on reverse-phase high-performance liquid chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of bacteriocin DT24 was approximately 7-kDa protein. The peptide is inactivated by proteolytic enzymes, trypsin and lipase but not when treated with catalase, α-amylase and pepsin. It showed bacteriostatic mode of action against uropathogenic E. coli. Such characteristics indicate that this bacteriocin-producing probiotic may be a potential candidate for alternative agents to control urinary tract infections and other pathogens.     

Complexes of Neutralizing and Non-Neutralizing Affinity Matured Fabs with a Mimetic of the Internal Trimeric Coiled-Coil of HIV-1 gp41

A series of mini-antibodies (monovalent and bivalent Fabs) targeting the conserved internal trimeric coiled-coil of the N-heptad repeat (N-HR) of HIV-1 gp41 has been previously constructed and reported. Crystal structures of two closely related monovalent Fabs, one (Fab 8066) broadly neutralizing across a wide panel of HIV-1 subtype B and C viruses, and the other (Fab 8062) non-neutralizing, representing the extremes of this series, were previously solved as complexes with 5-Helix, a gp41 pre-hairpin intermediate mimetic. Binding of these Fabs to covalently stabilized chimeric trimers of N-peptides of HIV-1 gp41 (named (CCIZN36)₃ or 3-H) has now been investigated using X-ray crystallography, cryo-electron microscopy, and a variety of biophysical methods. Crystal structures of the complexes between 3-H and Fab 8066 and Fab 8062 were determined at 2.8 and 3.0 Å resolution, respectively. Although the structures of the complexes with the neutralizing Fab 8066 and its non-neutralizing counterpart Fab 8062 were generally similar, small differences between them could be correlated with the biological properties of these antibodies. The conformations of the corresponding CDRs of each antibody in the complexes with 3-H and 5-Helix are very similar. The adaptation to a different target upon complex formation is predominantly achieved by changes in the structure of the trimer of N-HR helices, as well as by adjustment of the orientation of the Fab molecule relative to the N-HR in the complex, via rigid-body movement. The structural data presented here indicate that binding of three Fabs 8062 with high affinity requires more significant changes in the structure of the N-HR trimer compared to binding of Fab 8066. A comparative analysis of the structures of Fabs complexed to different gp41 intermediate mimetics allows further evaluation of biological relevance for generation of neutralizing antibodies, as well as provides novel structural insights into immunogen design.     

Inner Nuclear Layer Thickening Is Inversley Proportional to Retinal Ganglion Cell Loss in Optic Neuritis

Aim

To examine the relationship between retinal ganglion cell loss and changes in the inner nuclear layer (INL) in optic neuritis (ON).

Methods

36 multiple sclerosis (MS) patients with a history of ON and 36 age and sex-matched controls underwent Optical Coherence Tomography. The paramacular retinal nerve fiber layer (RNFL), combined ganglion cell and inner plexiform layers (GCL/IPL) and inner nuclear layer (INL) thickness were measured at 36 points around the fovea. To remove inter-subject variability, the difference in thickness of each layer between the ON and fellow eye of each patient was calculated. A topographic analysis was conducted.

Results

The INL of the ON patients was thicker than the controls (42.9µm versus 39.6µm, p=0.002). ON patients also had a thinner RNFL (27.8µm versus 32.2µm, p<0.001) and GCL/IPL (69.3µm versus 98.1µm, p<0.001). Among the controls, there was no correlation between RNFL and GCL/IPL as well as RNFL and INL, but a positive correlation was seen between GCL/IPL and INL (r=0.65, p<0.001). In the ON group, there was a positive correlation between RNFL and GCL/IPL (r=0.80, p<0.001) but a negative correlation between RNFL and INL (r=-0.61, p<0.001) as well as GCL/IPL and INL (r=-0.44, p=0.007). The negative correlation between GCL/IPL and INL strengthened in the ON group when inter-subject variability was removed (r=-0.75, p<0.001). Microcysts within the INL were present in 5 ON patients, mainly in the superior and infero-nasal paramacular regions. While patients with microcysts lay at the far end of the correlation curve between GCL/IPL and INL (i.e. larger INL and smaller GCL/IPL compared to other patients), their exclusion did not affect the correlation (r= -0.76, p<0.001).

Conclusions

INL enlargement in MS-related ON is associated with the severity of GCL loss. This is a continuous relationship and patients with INL microcysts may represent the extreme end of the scale.     

Screening of banana bunchy top virus through multiplex PCR approach

Bunchy top disease caused by the banana bunchy top virus (BBTV) is a serious disease in hill banana. Detection of the BBTV infection in the planting material could help in the effective management of the disease. An attempt was made to develop a sensitive polymerase chain reaction (PCR) and multiplex PCR-based method for detection of BBTV in hill banana. DNA was isolated from the experimental plants at third and sixth months after planting. Multiplex PCR was done with Coat Protein (CP) and Replicase (Rep) gene-specific primer, and banana ethylene insensitive like protein (EISL) primer as internal control to identify failure in PCR reaction. This study revealed that multiplex PCR is effective for BBTV screening in hill banana with the advantage of overcoming the false positive in PCR amplification.     

Insight into Actin Organization and Function in Cytokinesis from Analysis of Fission Yeast Mutants

Actin is a key cytoskeletal protein with multiple roles in cellular processes such as polarized growth, cytokinesis, endocytosis, and cell migration. Actin is present in all eukaryotes as highly dynamic filamentous structures, such as linear cables and branched filaments. Detailed investigation of the molecular role of actin in various processes has been hampered due to the multifunctionality of the protein and the lack of alleles defective in specific processes. The actin cytoskeleton of the fission yeast, Schizosaccharomyces pombe, has been extensively characterized and contains structures analogous to those in other cell types. In this study, primarily with the view to uncover actin function in cytokinesis, we generated a large bank of fission yeast actin mutants that affect the organization of distinct actin structures and/or discrete physiological functions of actin. Our screen identified 17 mutants with specific defects in cytokinesis. Some of these cytokinesis mutants helped in dissecting the function of specific actin structures during ring assembly. Further genetic analysis of some of these actin mutants revealed multiple genetic interactions with mutants previously known to affect the actomyosin ring assembly. We also characterize a mutant allele of actin that is suppressed upon overexpression of Cdc8p-tropomyosin, underscoring the utility of this mutant bank. Another 22 mutant alleles, defective in polarized growth and/or other functions of actin obtained from this screen, are also described in this article. This mutant bank should be a valuable resource to study the physiological and biochemical functions of actin.