Synthesis and biological activity of linear and cyclic enkephalins modified at the Gly3-Phe4 amide bond

As part of our continuing effort to define structure-activity relationships for enkephalin and design enzymatically resistant analogs, we report the synthesis and biological activities of linear and cyclic enkephalin analogs modified at the Gly3-Phe4 amide bond. The partial retro-inverso enkephalin analog Tyr-D-Ala-gGly-(R,S)-mPhe-Leu-NH2 and its cyclic counterpart, Tyr-cyclo[D-A2 bu-gGly-(R,S)-mPhe-Leu-], were synthesized as diastereomeric mixtures using solution methodology. The racemic benzylmalonate allowed the linear analog to be synthesized by fragment coupling at the reversed bond. Cyclization of the second analog was carried out at high concentration, eliminating formation of polymer by the use of an insoluble base. All gem-diaminoalkyl residues were prepared by conversion of peptidyl amides with benzene iodonium bis(trifluoroacetate). Diastereomers of both compounds were separable by reverse phase HPLC but those of the linear compound racemized rapidly under conditions of testing and were therefore tested together. All analogs tested had activities ranging from 6 to 14% of the activity of Leu enkephalin, indicating that the Gly3-Phe4 amide bond is important, though not crucial, for receptor binding.     

Über eine besondere Art von Laubfall bei einigen immergrünen Holzgewächsen

Ohne Zusammenfassung     

Enzymic synthesis,chemical characterisation and Sda activity of GalNAcß1-4[NeuAcα2-3]Galß1-4GlcNAc and GalNAcß1-4[NeuAcα2-3]Galß1-4Glc

The tetrasaccharides GalNAcß1-4[NeuAc2-3]Galß1-4Glc and GalNAcß1-4[NeuAc2-3]Galß1-4GlcNAc were synthesised by enzymic transfer of GalNAc from UDP-GalNAc to 3-sialyllactose (NeuAc2-3Galß1-4Glc) and 3-sialyl-N-acetyllactosamine (NeuAc2-3Galß1-4GlcNAc). The structures of the products were established by methylation and¹H-500 MHz NMR spectroscopy. In Sd^a serological tests the product formed with 3-sialyl-N-acetyllactosamine was highly active whereas that formed with 3-sialyllactose had only weak activity.     

Wheat dwarf virus, a geminivirus of graminaceous plants needs splicing for replication.

By analysing mRNAs with the polymerase chain reaction (PCR) and by studying in vitro generated mutants we have identified an intron in the genome of wheat dwarf virus (WDV), a geminivirus of cereals. Polypeptides whose expression is essential for the replication of the viral DNA have been defined. They are encoded by two distinct overlapping open reading frames (ORFs). The joining of these two ORFs by deletion of the intron as well as the introduction of a frameshift mutation within the intron do not prevent replication of the viral genome in suspension culture cells. In contrast to WDV, the geminiviruses of dicotyledonous plants possess a single continuous ORF, highly homologous to the two individual ones of WDV. We propose that mRNA splicing is a common feature of all geminiviruses of the Gramineae and might contribute to their host class specificity. The existence of a functional intron is a novel finding for the plant viruses.     

In vitro biological activities of the E6 and E7 genes vary among human papillomaviruses of different oncogenic potential.

Human papillomavirus type 16 (HPV-16) and HPV-18 are often detected in cervical carcinomas, while HPV-6, although frequently present in benign genital lesions, is only rarely present in cancers of the cervix. Therefore, infections with HPV-16 and HPV-18 are considered high risk and infection with HPV-6 is considered low risk. We found, by using a heterologous promoter system, that expression of the E7 transforming protein differs between high- and low-risk HPVs. In high-risk HPV-16, E7 is expressed from constructs containing the complete upstream E6 open reading frame. In contrast, HPV-6 E7 was efficiently translated only when E6 was deleted. By using clones in which the coding regions of HPV-6, HPV-16, and HPV-18 E7s were preceded by identical leader sequences, we found that the ability of the E7 gene products to induce anchorage-independent growth in rodent fibroblasts correlated directly with the oncogenic association of the HPV types. By using an immortalization assay of normal human keratinocytes that requires complementation of E6 and E7, we found that both E6 and E7 of HPV-18 could complement the corresponding gene from HPV-16. However, neither E6 nor E7 from HPV-6 was able to substitute for the corresponding gene of HPV-16 or HPV-18. Our results suggest that multiple factors, including lower intrinsic biological activity of E6 and E7 and differences in the regulation of their expression, account for the low activity of HPV-6, in comparison with HPV-16 and HPV-18, in in vitro assays. These same factors may, in part, account for the apparent difference in oncogenic potential between these viruses.     

Structure-activity relationships of cyclic β-casomorphin-5 analogues

Cyclic analogues of the β-casein-derived opioid peptide β-casomorphin-5 (H-Tyr-Pro-Phe-Pro-Gly-OH) were prepared through substitution of the Pro² residue with various ,ω-diamino acid residues (lysine, ornithine, 2,4-diaminobutyric acid) and cyclization of the ω-amino group to the C-terminal carboxyl function. Compounds of this type, with D-configuration at the 2-position residue, showed high opioid receptor affinity with some preference for μ receptors over δ receptors, high potency in the guinea pig ileum assay and considerable activity in the mouse vas deferens assay. Configurational inversion at the 4-position in these cyclic analogues resulted in enhanced affinity for both μ and δ receptors, whereas N-methylation of the Phe³ residue produced a potency decrease.     

Nonlinear increase of elongation rate of actin filaments with actin monomer concentration

The nonlinear increase of the elongation rate of actin filaments above the critical monomer concentration was investigated by nucleated polymerization of actin. Significant deviations from linearity were observed when actin was polymerized in the presence of magnesium ions. When magnesium ions were replaced by potassium or calcium ions, no deviations from linearity could be detected. The nonlinearity was analyzed by two simple assembly mechanisms. In the first model, if the ATP hydrolysis by polymeric actin is approximately as fast as the incorporation of monomers into filaments, terminal subunits of lengthening filaments are expected to carry to some extent ADP. As ADP-containing subunits dissociate from the ends of actin filaments faster than ATP-containing subunits, the rate of elongation of actin filaments would be nonlinearly correlated with the monomer concentration. In the second model (conformational change model), actin monomers and filament subunits were assumed to occur in two conformations. The association and dissociation rates of actin molecules in the two conformations were thought to be different. The equilibrium distribution between the two conformations was assumed to be different for monomers and filament subunits. The ATP hydrolysis was thought to lag behind polymerization and conformational change. As under the experimental conditions the rate of ATP hydrolysis by polymeric actin was independent of the concentration of filament ends, the observed nonlinear increase of the rate of elongation with the monomer concentration above the critical monomer concentration was unlikely to be caused by ATP hydrolysis at the terminal subunits. The conformational change model turned out to be the simplest assembly mechanism by which all available experimental data could be explained.