Functional drug targeting to erythrocytes in vivo using antibody bearing liposomes as drug vehicles

Covalent attachment of anti-erythrocyte F(ab')2 to the liposome surface has recently been shown to considerably enhance the liposome binding to erythrocytes in vivo. These antibody bearing liposomes have now been found quite effective as vehicles for delivering the antimalarial drug, chloroquine, to erythrocytes in Plasmodium berghei-infected mice. This demonstrates the usefulness of antibody targeted liposomes as carriers for site-specific drug delivery.     

Bioenergetic consequences of protein overexpression in Saccharomyces cerevisiae

Summary Some bioenergetic consequences of overexpression of plasmid-encoded homologous (phosphoglycerate kinase), and heterologous (prochymosin), protein in S. cerevisiae strains grown in chemostat culture have been investigated. Both overexpressing strains were found to exhibit similar fermentation patterns despite a 10-fold difference in product expression levels. Biomass yields were lower than those for a control strain, and the onset of oxido-fermentative metabolism occurred at a lower dilution rate. A marked rise in cellular ATP content with increasing dilution rate during oxidative growth was observed in the strain overexpressing yeast phosphoglycerate kinase (PGK); this at present cannot be adequately explained. The inorganic phosphate content of the overexpressing strains was higher than that of the control and the phosphorylation potential of the prochymosin expressing strain was up to 10-fold lower than both the control and PGK overexpressing strains. It is proposed that expression of heterologous prochymosin imposes a greater energy drain on the host than overexpression of homologous PGK. This energetic drain may be a limiting factor in heterologous gene expression.     

Increased therapeutic efficacy of zidovudine in combination with vitamin E

Antiviral activity and bone marrow toxicity of 3'-azido-3'deoxythymidine (Zidovudine; AZT) was evaluated in the presence of alpha-D-tocopherol acid succinate (ATS) in the MT4 cell line and in murine hematopoietic progenitor cells, respectively. At varying concentrations (.016 to .125 microM) of AZT, addition of ATS (5 to 15 micrograms/ml) showed a dose-dependent increase in anti-HIV activity. The ED90 of AZT in this test system was 0.37 microM, whereas in the presence of ATS (15 micrograms/ml) it was 0.06 microM, thus producing an approximately 6-fold increase in anti-HIV activity. In contrast, in murine bone marrow cells, ATS (4 micrograms/ml) showed significant protection (p less than 0.05) against AZT-induced toxicity as measured by CFU-E and CFU-GM assays. The IC50 values in the presence and absence of ATS for CFU-E were 3.7 and 1.5 microM, whereas for CFU-GM were 6.0 and 2.7 microM, respectively. Overall, these data suggest that AZT in combination with ATS has greater therapeutic efficacy against HIV-1.     

A pro-drug of zidovudine with enhanced efficacy against human immunodeficiency virus

In an attempt to alleviate the drug-related toxicity of zidovudine in patients with AIDS, a pro-drug of zidovudine, 5'-[(1,4-dihydro-1-methyl-3-pyridinylcarbonyl)oxy]-3'-azido-2',3'- dideoxythymidine (DP-AZT), has been evaluated. Cellular uptake by H9 cells and peripheral blood lymphocytes (PBL) with zidovudine and DP-AZT showed at least a 50% greater intracellular concentration of DP-AZT within 2 hr. DP-AZT was significantly less toxic to murine bone marrow cells as measured by CFU-E assay. The ED50 concentration to inhibit the production of HIV specific p24 antigen was 0.05 microM for DP-AZT whereas zidovudine required 0.125 microM. These results demonstrated that DP-AZT has a higher therapeutic ratio than zidovudine as an anti-HIV-1 agent.     

Cleavage of the PO glycoprotein of the rat peripheral nerve myelin: Tentative identification of cleavage site and evidence for the precursor-product relationship

The incubation of sciatic nerve slices in Krebs Ringer bicarbonate (KRB) buffer (pH 7.4) at 37°C, or the incubation of freshly isolated myelin in ammonium bicarbonate buffer (pH 8), resulted in the generation of a 24kDa protein with a concomitant decrease of PO protein. The conversion of PO into 24kDa protein was blocked by heating isolated myelin at 100°C for 5 min suggesting that the reaction is enzyme mediated. Inclusion of the protease inhibitors and chelating agent to isolated myelin did not prevent the formation of 24kDa protein. Similarly, addition of CaCl₂ to isolated myelin did not accentuate the formation of 24kDa protein suggesting that the conversion of PO into 24kDa protein may not be due to Ca²⁺ activated protease. It is postulated that the formation of 24kDa protein may be due to neutral protease and/or metalloproteinase associated with the PNS myelin. 24kDa protein was purified and characterized. The N-terminal sequence of 1–17 amino acid residues of 24kDa protein was identical to PO. 24kDa protein was immunostained and immunoprecipitated with anti-PO antiserum indicating the immunological similarities between PO and 24kDa protein. Labeling of 24kDa protein with [³⁵S]methionine provided evidence that PO may be in all probability cleaved between Met-168 and Met-193. Further studies were carried out to demonstrate that 24kDa protein was phosphorylated, glycosylated and acylated like PO. Phosphorylation of 24kDa protein in the nerve slices was increased five-fold by phorbol esters and phosphoserine was the only phosphoamino acid identified after partial acid hydrolysis of 24kDa protein. These results suggested that serine residue phosphorylated by protein kinase C may be located in amino acid residues 1-168. 24kDa protein was stained with periodic Schiff reagent. In addition, 24kDa protein was fucosylated and the fucosylation of 24kDa protein was inhibited (70%) by tunicamycin, providing evidence that it is N-glycosylated. Recently, it was demonstrated that both PO and 24kDa protein were fatty acylated with [³H]palmitic acid in the nerve slices and fatty acids are covalently linked to these proteins (Agrawal, H.C. and Agrawal, D. 1989, Biochem. J. 263:173–177). The time course of inhibition of acylation by cycloheximide of 24kDa protein was identical to PO. Cycloheximide inhibited acylation of PO and 24kDa protein by 61% and 58% respectively, whereas, monensin had little affect on the fatty acylation of these proteins. Less [³H]palmitic acid and¹⁴C-amino acids were incorporated into 24kDa protein when compared to PO between 5–30 min after incubation of the nerve slices. However, more radioactivity was incorporated into 24kDa protein after 60 min when compared to PO under identical conditions. These results provided evidence of a precursor-product relationship between PO and 24kDa protein. Therefore, PO may be cleaved into 24kDa protein in the myelin membrane following its acylation and glycosylation in the Schwann cells.     

Analysis of nuclear proteins from silk glands ofBombyx mori

A gentle method for the isolation of nuclei from developing silk glands ofBombyx mori has been standardized. The nuclei, whether isolated or directly visualizedin situ within the silk glands, exhibit complex morphology. The nuclei occupy almost the entire volume of the gigantic silk gland cells. Although the isolated nuclei still retain their ramified morphology, being polyploid they are fragile and often become fragmented. The histone and low-salt-extractable proteins from nuclei isolated from the middle and posterior silk glands on different days of the fourth and fifth instars of larval development have been analysed. The histones did not show any stage- or tissue-specific variations whereas the low-salt-extractable proteins showed some developmental stage specific variation. Using the antibody raised against one such protein, its absence in the early stage of development has been confirmed by Western blotting techniques. This developmental stage specific protein may be functionally linked to some activities responsible for boosting up the production of silk or silk-related proteins during the fifth instar of larval development.     

Cytochrome c: ion binding and redox properties. Studies on ferri and ferro forms of horse, bovine, and tuna cytochrome c

The ion binding properties of horse, bovine, and tuna cytochrome c (both oxidized and reduced) have been measured using a combination of ultrafiltration, neutron activation, and ion chromatography. The ions investigated were chloride, phosphate, and Tris-cacodylate. Ion chromatography and neutron activation analysis techniques were employed to determine the concentration of free anions. Binding constants are obtained from modified Scatchard plots (in the range of 10-2000 M-1). The redox potentials for cytochrome c at different ionic strengths, pH 7.0, have been determined. In this paper we report the ionic strength and ion binding effects on the redox properties of horse, bovine, and tuna cytochrome c. Potential versus ionic strength dependence for horse, bovine, and tuna cytochrome c from the experimental data were compared with a theoretical model.     

Enhanced degradation of gamma-irradiated lignocelluloses by a new xylanolytic Flavobacterium sp. isolated from soil

An extracellular chitosanase produced by Rhodotorula gracilis CFR-1 that catalyses a limited degradation of chitosan with no detectable generation of glucosamine or reducing groups was identified. Ultracentrifugation, polyacrylamide gel electrophoresis and gel permeation studies suggest that chitosan of average molecular mass 36000 Da was reduced by the enzymic catalysis to nearly one-fourth this size without further hydrolysis of the products. The enzyme, produced constitutively by this yeast, was partially purified and some of its properties were studied.