A hingeless Fc fusion system for site-specific cleavage by IdeS

Fusion of proteins to the Fc region of IgG is widely used to express cellular receptors and other extracellular proteins, but cleavage of the fusion partner is sometimes required for downstream applications. Immunoglobulin G-degrading enzyme of Streptococcus pyogenes (IdeS) is a protease with exquisite specificity for human IgG, and it can also cleave Fc-fusion proteins at a single site in the N-terminal region of the CH2 domain. However, the site of IdeS cleavage results in the disulfide-linked hinge region partitioning with the released protein, complicating downstream usage of the cleaved product. To tailor the Fc fragment for release of partner proteins by IdeS treatment, we investigated the effect of deleting regions of IgG-derived sequence that are upstream of the cleavage site. Elimination of the IgG-derived hinge sequence along with several residues of the CH2 domain had negligible effects on expression and purity of the fusion protein, while retaining efficient processing by IdeS. An optimal Fc fragment comprising residues 235–447 of the human IgG1 heavy chain sufficed for efficient production of fusion proteins and minimized the amount of residual Ig-derived sequence on the cleavage product following IdeS treatment. Pairing of this truncated Fc fragment with IdeS cleavage enables highly specific cleavage of Fc-fusion proteins, thus eliminating the need to engineer extraneous cleavage sequences. This system should be helpful for producing Fc-fusion proteins requiring downstream cleavage, particularly those that are sensitive to internal miscleavage if treated with alternative proteases.     

Direct evidence for Ca2+ regulation of hyphal branch induction

Grinberg, A., and Heath, I. B. 1997. Direct evidence for Ca2+ regulation of hyphal branch induction. 22, 127-139. Irradiation of growing hyphae of Saprolegnia ferax with microbeams of UV (300-380 and 385-450 nm) light induced an increase in cytoplasmic [Ca2+] followed by precocious formation of one or more branches within about 4 min. The distribution of branches was strongly skewed toward the subapical side of the irradiation site, but otherwise was apparantly random. Apical (10-&mgr;m) irradiations were more effective than subapical (50-&mgr;m) ones in that they induced branches at comparable frequencies but with lower doses, consistent with higher concentrations of putative target intracellular Ca2+ storage structures in this region. Once formed, induced adjacent branches seem to compete for "resources," with those closer than approximately 50 &mgr;m inhibiting each other. The results are most consistent with Ca2+-induced accumulation of branch initiating factors being the cause, not the consequence, of branch formation, thus supporting a primary role for Ca2+ in regulation of hyphal tip growth. Copyright 1997 Academic Press. Copyright 1997 Academic Press     

Synthesis of acridine derivatives of amino acid hydrazines and their antimalarial activity

2-Methoxy-6-chloroacridine-9-yl- and 2-ethoxy-6-nitroacridine-9-yl-hydrazides of glycine, alpha- and beta-alanines, gamma-aminobutiric acid, epsilon-aminocaproic acid have been synthesized and their antimalarial activity has been investigated. The compounds were found to inhibit the growth of malaria parasite P. falciparum in in vitro cultures. Fifty per cent inhibitory concentrations ranged from 2 x 10(-7) to 6 x 10(-7) M and corresponded to therapeutic concentrations of known quinoline and acridine antimalarial drugs. The beta-alanine and gamma-aminobutiric acid derivatives were the most active and showed high activity against a chloroquine resistant strain of P. falciparum.     

Characterization of B278, a phage different from Mu that also produces auxotrophic mutations in Escherichia coli K12

Bacteriophage B278 has been characterized and compared with Mu, the only phage known to produce random mutations in E. coli. Although both phages are morphologically indistinguishable and have a similar host range, they clearly differ at both the protein and the DNA level. B278 apparently possesses a DNA protection mechanism that is different from the mom system described for Mu.     

Collagen and fibronectin synthesis by trisomic and triploid fibroblasts from human spontaneous abortuses

Summary Collagen and fibronectin synthesis by trisomic and triploid fibroblasts derived from human spontaneous abortuses was studied. It was demonstrated that the level of fibronectin and collagen production in fibroblasts with trisomy 7, trisomy 9, and triploidy was reduced as compared with diploid cells. A correlation between this observation and an increased rate of intracellular ¹⁴C-procollagen degradation was also established for the anomalous strains. No difference in hydroxylation of ¹⁴C-proline residues in 1() and 2() collagen chains and no fluctuation in the collagen type (): type ratio was found in the strains with the abnormal karyotypes. It was concluded that differentiation of the abnormal fibroblasts was impaired. The data also favour the hypothesis that the deficiency of the fibroblasts in producing proteins may account for a variety of anatomic abnormalities of embryos.     

Activation of morphogenesis and proliferation by low specificity chemical effectors

Activation of highly specific biochemical processes by simple chemical agents is demonstrated for morphogenesis (anlage and development of female gametophyte in cereal) and mitosis (in cell cultures and animal and plant tissues). The effects of these agents are tissue-specific. Structure--activity relationship is analyzed in this group of compounds. Thus, the phenomenon reveals the exact pathways of the influence of allelopathic and anthropogenic chemical agents on evolution of plant biocenoses.     

Organization of fusicoccin receptor in higher plant plasma membrane: relationship between affinity and molecular mass

Higher plant plasma membranes carry receptors of different affinity for the phytotoxin fusicoccin. Reception of fusicoccin involves proteins belonging to the highly conserved 14-3-3 family, but the complete structure of the fusicoccin receptor (FCR) is unknown. Using radiation inactivation analysis, we estimated the molecular masses of low-affinity and high-affinity FCR at 63 +/- 7 and 130 +/- 15 kD, respectively. The dose dependences of receptor inactivation indicate that microsomal specimens contain "silent" FCRs of 420 +/- 90 kD in amounts commensurate with that of the active FCRs. Both low- and high-affinity FCRs are inactivated by hydrolytic enzymes from the outer surface of the plasma membrane, and impairment of protoplast integrity causes an irreversible transition of the low-affinity binding site into the high-affinity one. A scheme is proposed for the organization of different types of FCR in the plasma membrane, implying that the membrane affinity for fusicoccin reflects the interaction between proteins in the FCR complex.     

Enzymes of cultured human fibroblasts. IV. Enzyme activity in trisomy C strains]

The activity of five enzymes (AIP, AcP, GOT, LDH, MDH) was investigated in four cell strains derived from spontaneous abortuses with C-trisomy (three cell strains with trisomy 7, one--with trisomy 9). Significant differences in the activity of three enzymes were revealed. In all the strains AIP activity was lower and GOT activity--higher than in diploid strains. Lowering of AcP level was found in three strains (two cell strains with trisomy 7, one--with trisomy 9). The data obtained are evaluated as a result of disturbed regulatory interrelations in an abnormal genome.