Raman spectra of ten aqueous transfer RNAs and 5S RNA. Conformational comparison with yeast phenylalanine transfer RNA.

Eleven native transfer RNAs have been prepared so as to maintain their Mg2+ content. Their aqueous Raman spectra show a high, relatively constant amount of order in the ribophosphate backbone, as indicated by the ratio 1.73 +/- 0.05 for I814/I1100 in all samples. Variation in the effectiveness of stacking of guanine and adenine bases is seen, though most of the transfer RNAs studied have a comparable degree of stacking to that found in phenylalanine transfer RNA from yeast, whose tertiary structure has been determined by X-ray crystallography. The spectrum of Escherichia coli 5S RNA indicates that the stacking efficiency of the guanine bases is much higher in 5S RNA than in yeast in phenylalanine transfer RNA, while that of the adenine bases is lower.     

A Proposal for the Adaptive Significance of the Development of the Lamprey Fat Column

Abstract Lampreys differ from the other extant agnathan group, the hagfishes, in possessing a fat column above the nerve cord. It is suggested that this region of fat is just one of many sites of lipid storage that have been developed to provide energy reserves for the lengthy non-trophic periods found during the life cycle of lampreys. The incorporation of this fat within an extension of the nerve cord sheath suggests that it could act as a protective dorsal cushion for the nerve cord, which would otherwise be more exposed due to the absence of complete vertebral arches. The development of the fat column as the principal blood cell forming structure in the adult can apparently be related to certain changes that take place during metamorphosis, particularly in the circulatory system.     

Perlecan domain 1 recombinant proteoglycan augments BMP-2 activity and osteogenesis

ABSTRACT: BACKGROUND: Many growth factors, such as bone morphogenetic protein (BMP)-2, have been shown to interact with polymers of sulfated disacharrides known as heparan sulfate (HS) glycosaminoglycans (GAGs), which are found on matrix and cell-surface proteoglycans throughout the body. HS GAGs, and some more highly sulfated forms of chondroitin sulfate (CS), regulate cell function by serving as co-factors, or co-receptors, in GF interactions with their receptors, and HS or CS GAGs have been shown to be necessary for inducing signaling and GF activity, even in the osteogenic lineage. Unlike recombinant proteins, however, HS and CS GAGs are quite heterogenous due, in large part, to post-translational addition, then removal, of sulfate groups to various positions along the GAG polymer. We have, therefore, investigated whether it would be feasible to deliver a DNA pro-drug to generate a soluble HS/CS proteoglycan in situ that would augment the activity of growth-factors, including BMP-2, in vivo. RESULTS: Utilizing a purified recombinant human perlecan domain 1 (rhPln.D1) expressed from HEK 293 cells with HS and CS GAGs, tight binding and dose-enhancement of rhBMP-2 activity was demonstrated in vitro. In vitro, the expressed rhPln.D1 was characterized by modification with sulfated HS and CS GAGs. Dose-enhancement of rhBMP-2 by a pln.D1 expression plasmid delivered together as a lyophilized single-phase on a particulate tricalcium phosphate scaffold for 6 or more weeks generated up to 9 fold more bone volume de novo on the maxillary ridge in a rat model than in control sites without the pln.D1 plasmid. Using a significantly lower BMP-2 dose, this combination provided more than 5 times as much maxillary ridge augmentation and greater density than rhBMP-2 delivered on a collagen sponge (InFuse[trade mark sign]). CONCLUSIONS: A recombinant HS/CS PG interacted strongly and functionally with BMP-2 in binding and cell-based assays, and, in vivo, the pln.247 expression plasmid significantly improved the dose-effectiveness of BMP-2 osteogenic activity for in vivo de novo bone generation when delivered together on a scaffold as a single-phase. The use of HS/CS PGs may be useful to augment GF therapeutics, and a plasmid-based approach has been shown here to be highly effective.     

Protein Kinase C-δ Associates with Vimentin Intermediate Filaments in Differentiated HL60 Cells

The subcellular localization of protein kinase C (PKC)-δ was determined in HL60 cells differentiated toward monocytes/macrophages by treatment with TPA. PKC-δ was detected in the nucleus and cytoplasm of differentiated HL60 cells and, more specifically, associated with structures resembling intermediate filaments. Indirect immunostaining revealed that PKC-δ colocalized with vimentin in the cytosol and perinuclear region of these cells. Immunoprecipitation studies showed that PKC-δ was in an active (autophosphorylated) state in differentiated HL60 cells and that vimentin immunoprecipitated from these cells was also phosphorylated. Treatment of HL60 cells with the PKC-specific inhibitor chelerythrine decreased the phosphorylation of vimentin. These data suggest that vimentin is a substrate for PKC-δ and that this PKC isoenzyme may play a specific role in the regulation of shape change and cell adhesion during HL60 differentiation.