4-N-Alkanoyl and 4-N-alkyl gemcitabine analogues with NOTA chelators for 68-gallium labelling

The conjugation of 4-N-(3-aminopropanyl)-2′-deoxy-2′,2′-difluorocytidine with 2-(p-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (SCN-Bn-NOTA) ligand in 0.1?M Na₂CO₃ buffer (pH 11) at ambient temperature provided 4-N-alkylgemcitabine-NOTA chelator. Incubation of latter with excess of gallium(III) chloride (GaCl₃) (0.6?N AcONa/H₂O, pH?=?9.3) over 15?min gave gallium 4-N-alkylgemcitabine-NOTA complex which was characterized by HRMS. Analogous [⁶⁸Ga]-complexation of 4-N-alkylgemcitabine-NOTA conjugate proceeded with high labeling efficiency (94%–96%) with the radioligand almost exclusively found in the aqueous layer (～95%). The high polarity of the gallium 4-N-alkylgemctiabine-NOTA complex resulted in rapid renal clearance of the ⁶⁸Ga-labelled radioligand in BALB/c mice.     

Endothelial cell junctions

In the course of a freeze-cleave study on intercellular junctions in the regenerating rat liver, we observed an unusual array of intramembranous particles located in regions of contact between endothelial cells lining the hepatic sinusoids. These arrays were characterized by an accumulation of particles which resembled a zonula occludens in their linear deployment but differed in that the contact regions were composed of individual particles which remained separated from each other by regular particle-free intervals.     

Presentation of two epitopes of the preS2 region of hepatitis B virus on live recombinant bacteria

Having developed a genetic procedure to expose a foreign epitope at the surface of Escherichia coli by using the outer membrane LamB protein as a carrier, we apply this procedure to express two distinct portions of the preS2 region of hepatitis B virus: region A, residues 132-145, and region B, residues 153-171. The resulting hybrid proteins (LamB-preS2 A and LamB-preS2 B) were normally expressed, stable, and still kept most biologic functions of LamB. The corresponding bacterial strains were used directly as immunogens in rabbits and mice. Both viral sequences were found to be immunogenic in the two animal species. With LamB-preS2 A, antibodies induced were able to react with the viral particles and the immobilized peptide. With LamB-preS2 B, the antibodies raised were not able to recognize the immobilized peptide. However, the results suggest that the B epitope, inserted in LamB, was at least as efficient as the corresponding synthetic peptide in raising antiviral antibodies. Thus, epitope presentation with LamB may present advantages for immunization. We also have shown that peptide A is an essential part of the polymerized human serum albumin receptor. These results, which validate further the LamB vector system for epitope presentation, provide information on the two hepatitis B regions expressed.     

Rat liver HMG1: a physiological nucleosome assembly factor.

Incubation of rat liver single-stranded DNA-binding protein HMG1 with the four core histones at 0.15 M NaCl favors histone association primarily into tetramers and, to a lesser extent, into octamers. The assembly of pre-formed histone-HMG1 complexes with DNA yields nucleosome-like subunits which satisfy most of the criteria defining native core particles: (i) the circular DNA extracted from the complexes is supercoiled indicating that the initially relaxed DNA acquired superhelical turns during complex formation in the presence of topoisomerase I; (ii) the digestion of the complexes with micrococcal nuclease yields a DNA fragment of approximately 140 bp in length; (iii) electron microscopy of the reconstituted complexes shows a beaded structure with the DNA wrapped around the histone cores, leading to a reduction in the contour length of the genome compared with free DNA. Moreover, in the presence of HMG1, nucleosome assembly occurs rapidly at 0.15 M NaCl. Therefore, in addition to its DNA-binding properties, HMG1 mediates the assembly of nucleosomes in vitro under conditions of physiological ionic strength. The possible involvement of these properties in the DNA replication process is discussed.     

Ca2+-independent binding and cellular expression profiles question a significant role of calmyrin in transduction of Ca2+-signals to Alzheimer's disease-related presenilin 2 in forebrain

The interaction between the EF-hand Ca(2+)-binding protein calmyrin and presenilin 2 (PS2) has been suggested to play a role in Alzheimer's disease (AD). We now report that calmyrin binds specifically endogenous PS2 and not PS1. However, binding appears to be Ca(2+)-independent and calmyrin does not exhibit a Ca(2+)-dependent translocation to intracellular membranes as demonstrated in a Ca(2+)-myristoyl switch assay. Moreover, calmyrin is only present at very low levels in brain areas associated with the onset of AD. In rat, forebrain calmyrin is localized only in a subset of principal neurons, similarly as in human forebrain. Finally, subcellular fractionation demonstrates only a limited overlap of calmyrin and PS2 at neuronal membranes. We therefore conclude that calmyrin will not contribute significantly as a Ca(2+)-sensor that transduces Ca(2+)-signaling events to PS2 in forebrain.