The Locational Impact of Site-Specific PEGylation: Streamlined Screening with Cell-Free Protein Expression and Coarse-Grain Simulation.

Although polyethylene glycol (PEG) is commonly used to improve protein stability and therapeutic efficacy, the optimal location for attaching PEG onto proteins is not well understood. Here, we present a cell-free protein synthesis-based screening platform that facilitates site-specific PEGylation and efficient evaluation of PEG attachment efficiency, thermal stability, and activity for different variants of PEGylated T4 lysozyme, including a di-PEGylated variant. We also report developing a computationally efficient coarse-grain simulation model as a potential tool to narrow experimental screening candidates. We use this simulation method as a novel tool to evaluate the locational impact of PEGylation. Using this screen, we also evaluated the predictive impact of PEGylation site solvent accessibility, conjugation site structure, PEG size, and double PEGylation. Our findings indicate that PEGylation efficiency, protein stability, and protein activity varied considerably with PEGylation site, variations that were not well predicted by common PEGylation guidelines. Overall our results suggest current guidelines are insufficiently predictive, highlighting the need for experimental and simulation screening systems such as the one presented here.

Direct toxic effects of immunopotentiators on monocytic, myelomonocytic, and histiocytic or macrophage tumor cells in culture.

Four murine monocyte, myelomonocyte, and histiocyte or macrophage tumor cell lines adapted to culture were growth inhibited by one or more of the following macrophage-activating substances: Mycobacterium bovis, Bacillus Calmette-Guérin strain, zymosan, lipopolysaccharide, and dextran sulfate, as well as tuberculin purified protein derivative, but not latex beads. Lipopolysaccharide was effective with one line at 4 ng/ml. All four lines actively phagocytosed zymosan and latex beads. In many cases the growth inhibition was apparently immediate but only cytostatic, and cell proliferation resumed upon removal of the drug. Bacillus Calmette-Guérin, live or boiled, was toxic to some of the tumor lines. Synthesis of lysozyme by all the cell lines in the monocyte series and production of granulocyte colony-stimulating factor by the myelomonocytic leukemia were not inhibited during several days of zero growth conditions in the presence of drugs. Since these agents had no direct effect on other hematopoietic tumor types (myeloma, T-lymphoma, mastocytoma) at the same or up to 10(4) higher concentrations, it is proposed that the sensitive tumors retain specific receptors for immunostimulants, either at the cell surface or within the cell in the case of phagocytosable particles. The binding of these agents to physiological receptors leads to stimulation and mitogenesis in normal macrophages and lymphocytes but leads to growth inhibition without affecting differenetiated functions in the corresponding tumor lines.

Primary cultures of human blood-born macrophages grown on hydrophobic teflon membranes.

Human blood-born monocytes have been cultivated on the hydrophobic side of Teflon foils (fluorinated ethylene propylene copolymer) using human pooled AB-serum as essential growth factor. At any stage of culture these in vitro maturing macrophages can easily be detached from the Teflon membrane and subjected to further experimentation. Once established, primary macrophage cultures can be maintained in medium containing 10% FCS for up to 3 months. Lysozyme secretion increased more than 10-fold during the sequential process of monocyte transformation into macrophages and correlates with cell number and stage of maturation. The ability to inhibit growth of human permanent tumor cell lines also developed during macrophage maturation. Studies on the cells of 22 healthy donors revealed a reproducible activity of mature macrophages against K562 and MOLT4 tumor cells. Our system will facilitate investigations on various aspects of human macrophage differentiation and function.

Influence of soluble suture factors on in vitro macrophage function.

Suture materials may interact with immune competent cells and thereby affect localized immunity. Macrophages are central to the inflammatory response and coordinate wound healing. They are also involved in the clearance of foreign material, bacteria and malignant cells. We studied the influence of soluble factors associated with silk, steel, nylon, polyglactin, polydioxanone and chromic catgut sutures on macrophage adherence, phagocytosis and the production of lysozyme and tumour necrosis factor. Soluble factors from suture materials influenced macrophage behaviour in vitro causing cellular activation, functional impairment and alterations in secreted levels of the cytokine tumour necrosis factor and the bactericidal agent lysozyme. Of the six materials studied, polyglactin had the most extreme effect, causing significant inhibition of cell adherence and lysozyme production. Silk also exerted a considerable effect on macrophages, significantly inhibiting adherence. In contrast, steel and polydioxanone media caused minimal inhibition of macrophage function although, as with all materials, they did activate the cells. This study has demonstrated that sutures release immunotoxic factors which considerably influence macrophage behaviour in vitro. These effects may have important clinical implications.

Synthesis of a regioregular zwitterionic conjugated oligoelectrolyte, usable as an optical probe for detection of amyloid fibril formation at acidic pH.

Changes of the optical properties of conjugated polyelectrolytes have been utilized to monitor noncovalent interactions between biomolecules and the conjugated polyelectrolytes in sensor applications. A regioregular, zwitterionic conjugated oligoelectrolyte was synthesized in order to create a probe with a defined set of optical properties and hereby facilitate interpretation of biomolecule-oligoelectrolyte interactions. The synthesized oligoelectrolyte was used at acidic pH as a novel optical probe to detect amyloid fibril formation of bovine insulin and chicken lysozyme. Interaction of the probe with formed amyloid fibrils results in changes of the geometry and the electronic structure of the oligoelectrolyte chains, which were monitored with absorption and emission spectroscopy.

[Lysozyme content in the blood serum and saliva of carriers of pethogenic staphylococci]. / Soderzhanie lizotsima v syvorotke krovi i v sliun nositelei patogennykh stafilokokkov

90 serum and 273 saliva samples from pathogenic staphylococci carriers and healthy persons (control group) were studied. It was found that the state of pathogenic staphylococci carrying on the nose mucosa had no significant effect on the lysozyme levels in the blood serum, while assisted an increase in the content of the substance in the saliva. Increased lysozyme levels in the saliva of the pathogenic staphylococci carriers showed no clear connections with lysozyme production by the pathogens.

Expression profile of human defensins and antimicrobial proteins in oral tissues.

Antimicrobial peptides and proteins are an important part of the innate host defense. In the present study, the expression profile of three human alpha-defensins, of two human beta-defensins (hBD) and of phospholipase A-2 (PLA-2) and lysozyme was determined by reverse transcription-polymerase chain reaction (RT-PCR) in 56 non-inflamed and 18 inflamed oral tissue samples and primary oral keratinocytes and fibroblasts. The transcripts for hBD-1 and -2 as well as for PLA-2 and lysozyme were found to be widely expressed. In the group of the alpha-defensins, the message for the human neutrophil peptide-1 (HNP-1) was frequently detected, whereas an expression of human Paneth's cell defensin-5 (HD-5) was identified in only a minority of samples. Transcripts for HD-6 were not detectable in any sample. Oral keratinocytes but not fibroblasts contained transcripts for the beta-defensins, suggesting that these defensins are produced in the epithelial compartment. In contrast, mRNA expression of neutrophil-derived HNP-1 and PLA-2 was not observed in any of these cells. These results suggest an important role for hBD-1 and hBD-2 in the innate oral epithelial host defense.

Virulence factors of biotypes of Staphylococcus epidermidis from clinical sources.

The biotyping scheme of Baird-Parker was applied to cultures of Staphylococcus epidermidis from patients. In all, 63.6% of 228 cultures belonged to biotype 1, followed by biotypes 4, 3, and 2 in decreasing order of incidence. When classified according to clinical source of isolation, cultures of S. epidermidis were most frequently isolated from urine, with 39.5% of 228 cultures from this source. Each of the four biotypes was distributed throughout all nine catagories of clinical sources. The production of virulence factors was based on the results of three groups of tests: (i) deoxyribonuclease, urease, gelatinase, caseinase, and lysozyme production; (ii) lipolytic activity on the tweens; and (iii) hemolysin production. Enzymatic activity was highest for organisms in biotypes 1, followed by biotypes 3, 4, and 2 in decreasing order. Of the 228 cultures, 76.3% were lysed by lysostaphin. Resistance to antibiotics was highest for tetracycline, ampicillin, and penicillin, with rates of 54.8, 69.3, and 81.6%, respectively. The role of S. epidermidis as an etiological agent was studied by analyzing the laboratory and clinical data of 80 patients selected at random with bacteriuric S. epidermidis. Organisms in biotype 1 were most commonly associated with urinary tract infection. The significance of certain biotypes of S. epidermidis as opportunistic pathogens among compromised hosts in a hospital environment is discussed.

Enhanced bactericidal action of lysozyme to Escherichia coli by inserting a hydrophobic pentapeptide into its C terminus.

The mechanism of the enhanced bactericidal action to Escherichia coli of the lysozyme having a hydrophobic pentapeptide (Phe-Phe-Val-Ala-Pro) at its C terminus was investigated. The modified lysozyme, hydrophobic pentapeptide-fused lysozyme (HLz), was secreted in the culture medium from yeast harboring the expression plasmid, in which a synthetic DNA fragment encoding a hydrophobic pentapeptide was introduced to the 3'-end of the coding region of the lysozyme cDNA. Although CD analysis showed that HLZ was considerably different from wild-type lysozyme (WLz) in the secondary and tertiary structures, it retained 76% of the lytic activity of WLz. When E. coli cells were exposed to the WLz or HLz, the survival cells were significantly reduced only in the case of HLz. Periplasmic proteins from the HLz-treated cells were released to an extent similar to that from the WLz-treated cells, indicating that HLz has nearly the same action as WLz with respect to the disruption of the outer membrane and peptidoglycan. Experiments with E. coli phospholipid liposomes revealed that HLz dissipated the valinomycin-induced transmembrane electrochemical potential, but WLz did not. These results suggest that the enhanced bactericidal action of HLz to E. coli is due to disruption of the electrochemical potential of the inner membrane in cooperation with the inherent function of the lysozyme to the outer membrane and peptidoglycan.