A triple helix model for the structure of chromatin fiber

A model of chromatin fiber structure is presented in which a repeating unit of a trinucleosome forms a 3-dimensional zigzag. Twisting and compression of the zigzag result in a triple helix structure. The model is built mainly on the flow linear dichroism data showing that nucleosomal disc faces are tilted relative to the fiber axis, the orientation of nucleosomes does not change upon folding and unfolding of chromatin, and the orientation of nucleosomes is maintained by the globular domain of histone H1.     

Cell-mediated tumor-killing effect studied by using bilayer lipid membranes

The bilayer lipid membrane (BLM) system was used to investigate the tumor killing effect of natural killer (NK) cells under various experimental conditions. It was found that NK cells interact specifically with BLMs made from lipids and proteolipids isolated from target K562 cells inducing an increase of the membrane conductance. This effect was more pronounced when the NK cells were pretreated with interferon. A similar effect was observed when NK cells were pretreated with sodium selenite. The results suggest that changes in membrane conductance and permeability are involved in the mechanism of the tumor-killing effect mediated by NK cells.     

Ketamine esters and amides as short-acting anaesthetics: Structure-activity relationships for the side-chain

N-Aliphatic ester analogues of the non-opioid ketamine (1) retain effective anaesthetic/analgesic properties while minimising ketamine’s psychomimetic side-effects. We show that the anaesthetic/analgesic properties of these ester analogues depend critically on the length (from 2 to 4 carbons), polarity and steric cross-section of the aliphatic linker chain. More stable amide and ethylsulfone analogues generally showed weaker anaesthetic/analgesic activity. There was no correlation between the anaesthetic/analgesic properties of the compounds and their binding affinities for the N-methyl-d-aspartate (NMDA) receptor.     

Engineered Human Antibody Constant Domains with Increased Stability

The immunoglobulin (Ig) constant CH2 domain is critical for antibody effector functions. Isolated CH2 domains are promising as scaffolds for construction of libraries containing diverse binders that could also confer some effector functions. However, previous work has shown that an isolated murine CH2 domain is relatively unstable to thermally induced unfolding. To explore unfolding mechanisms of isolated human CH2 and increase its stability γ1 CH2 was cloned and a panel of cysteine mutants was constructed. Human γ1 CH2 unfolded at a higher temperature (T_m = 54.1 °C, as measured by circular dichroism) than that previously reported for a mouse CH2 (41 °C). One mutant (m01) was remarkably stable (T_m = 73.8 °C). Similar results were obtained by differential scanning calorimetry. This mutant was also significantly more stable than the wild-type CH2 against urea induced unfolding (50% unfolding at urea concentration of 6.8 m versus 4.2 m). The m01 was highly soluble and monomeric. The existence of the second disulfide bond in m01 and its correct position were demonstrated by mass spectrometry and nuclear magnetic resonance spectroscopy, respectively. The loops were on average more flexible than the framework in both CH2 and m01, and the overall secondary structure was not affected by the additional disulfide bond. These data suggest that a human CH2 domain is relatively stable to unfolding at physiological temperature, and that both CH2 and the highly stable mutant m01 are promising new scaffolds for the development of therapeutics against human diseases.Monoclonal antibodies (mAbs)² with high affinity and specificity are now well established therapeutics and invaluable tools for biological research. It appears that their use will continue to expand in both targets and disease indications. However, a fundamental problem for full-size mAbs that limits their applications is their poor penetration into tissues (e.g. solid tumors) and poor or absent binding to regions on the surface of some molecules (e.g. on the HIV envelope glycoprotein) that are accessible by molecules of smaller size. Antibody fragments, e.g. Fabs (∼60 kDa) or single chain Fv fragments (scFvs) (20∼30 kDa), are significantly smaller than full-size antibodies (∼150 kDa), and have been used as imaging reagents and candidate therapeutics. Even smaller fragments of antibodies are of great interest and advantageous for pharmaceutical applications including cancer targeting and imaging.During the last decade a large amount of work has been aimed at developing of small size binders with scaffolds based on various highly stable human and non-human molecules (1–8). A promising direction is the development of binders based on the heavy or light chain variable region of an antibody; these fragments ranging in size from 11 kDa to 15 kDa were called “domain antibodies” or “dAbs” (7, 9). A unique kind of antibodies composed only of heavy chains are naturally formed in camels, dromedaries, and llamas, and their variable regions can also recognize antigens as single domain fragments (10). Not only is the overall size of the dAbs much smaller than that of full-size antibodies but also their paratopes are concentrated over a smaller area so that the dAbs provide the capability of interacting with novel epitopes that are inaccessible to conventional antibodies or antibody fragments with paired light and heavy chain variable domains.The structure of the constant antibody domains is similar to that of the variable domains consisting of β-strands connected mostly with loops or short helices. The second domain of the α, δ, and γ heavy chain constant regions, CH2, is unique in that it exhibits very weak carbohydrate-mediated interchain protein-protein interactions in contrast to the extensive interchain interactions that occur between the other domains. The expression of murine CH2 in bacteria, which does not support glycosylation, results in a monomeric domain (11). It has been hypothesized that the CH2 domain (CH2 of IgG, IgA, and IgD, and CH3 of IgE and IgM) could be used as a scaffold and could offer additional advantages compared with those of dAbs because it contains binding sites or portions of binding sites conferring effector and stability functions (12).It was found previously that an isolated murine CH2 is relatively unstable at physiological temperature with a temperature of 50% unfolding (T_m) slightly higher than 37 °C (11). We have hypothesized that human CH2 would exhibit different stability because of significant differences in the sequence compared with its murine counterpart. Therefore, we have extensively characterized the stability of an isolated unglycosylated single CH2 domain. We found that its stability is significantly higher than the previously reported for the murine CH2. We further increased the stability of the human CH2 by engineering an additional disulfide bond between the A and G strands. One of the newly developed mutants, denoted as m01, exhibited significantly higher stability (T_m = 73.8 °C) than that of wild type CH2. We suggest that both the wild type CH2 and the newly developed mutant, m01, could be used as scaffolds for binders. These results also demonstrate for the first time that the stability of constant antibody domains can be dramatically increased by engineering of an additional disulfide bond. The increase in stability of isolated domains may result in an increase in stability of larger antibody fragments, e.g. Fc, and therefore could have implications as a general method for increasing antibody stability. Thus, it appears that further development of CH2 and its more stable variants as scaffolds could provide new opportunities for identification of potentially useful therapeutics.     

Structural details of monoclonal antibody m971 recognition of the membrane-proximal domain of CD22

Cluster of differentiation-22 (CD22) belongs to the sialic acid–binding immunoglobulin (Ig)-like lectin family of receptors that is expressed on the surface of B cells. It has been classified as an inhibitory coreceptor for the B-cell receptor because of its function in establishing a baseline level of B-cell inhibition. The restricted expression of CD22 on B cells and its inhibitory function make it an attractive target for B-cell depletion in cases of B-cell malignancies. Genetically modified T cells with chimeric antigen receptors (CARs) derived from the m971 antibody have shown promise when used as an immunotherapeutic agent against B-cell acute lymphoblastic leukemia. A key aspect of the efficacy of this CAR-T was its ability to target a membrane-proximal epitope on the CD22 extracellular domain; however, the molecular details of m971 recognition of CD22 have thus far remained elusive. Here, we report the crystal structure of the m971 fragment antigen-binding in complex with the two most membrane-proximal Ig-like domains of CD22 (CD22_d6–d7). The m971 epitope on CD22 resides at the most proximal Ig domain (d7) to the membrane, and the antibody paratope contains electrostatic surfaces compatible with interactions with phospholipid head groups. Together, our data identify molecular details underlying the successful transformation of an antibody epitope on CD22 into an effective CAR immunotherapeutic target.     

Glycan structures and antiviral effect of the structural subunit RvH2 of Rapana hemocyanin

Molluscan hemocyanins are very large biological macromolecules and they act as oxygen-transporting glycoproteins. Most of them are glycoproteins with molecular mass around 9000 kDa. The oligosaccharide structures of the structural subunit RvH2 of Rapana venosa hemocyanin (RvH) were studied by sequence analysis of glycans using MALDI-TOF-MS and tandem mass spectrometry on a Q-Trap mass spectrometer after enzymatical liberation of the N-glycans from the polypeptides. Our study revealed a highly heterogeneous mixture of glycans of the compositions Hex_0-9 HexNAc_2-4 Hex_0-3 Pent_0-3 Fuc_0-3. A novel type of N-glycan, with an internal fucose residue connecting one GalNAc(β1-2) and one hexuronic acid, was detected, as also occurs in subunit RvH1. A glycan with the same structure but with two deoxyhexose residues was observed as a doubly charged ion. Antiviral effects of the native molecules of RvH and also of Helix lucorum hemocyanin (HlH), of their structural subunits, and of the glycosylated functional unit RvH2-e and the non-glycosylated unit RvH2-c on HSV virus type 1 were investigated. Only glycosylated FU RvH2-e exhibits this antiviral activity. The carbohydrate chains of the FU are likely to interact with specific regions of glycoproteins of HSV, through van der Waals interactions in general or with certain amino acid residues in particular. Several clusters of these residues can be identified on the surface of RvH2-e.     

Dense and sparse aggregations in complex motion: Video coupled with simulation modeling

Investigations into the complex behaviors of aggregations of highly mobile animals have not used the link between image processing technology and simulation modeling fruitfully to address many fundamental ecological issues. Examples include population censusing, which remains difficult despite the demonstrated ecological importance of assessing abundance and density of organisms. We introduce a theoretical framework that connects thermal infrared video imaging with an individual-based simulation model—an approach that appears to be applicable to a diverse set of aggregated, highly mobile, nocturnal animals. To demonstrate the framework two applications are presented. The first is a dense aggregation of Brazilian free-tailed bats (Tadarida brasiliensis) that exhibits an emergence pattern that has complex dynamics and the second is a sparse local aggregation of agricultural pest moths whose dynamics are insipid. The first application uses individual-based modeling to mimic the behavior in the video of bats during a nightly emergence from a cave and to provide reliable estimates of the numbers, and associated error bounds. The second application uses video recordings of sparse aggregations to provide consistent estimates of the numbers of flying noctuid moths recorded over a corn and cotton-dominated agroecosystem in south-central Texas. This does not use a mathematical model because error estimates tend to be small.     

Human anti-plague monoclonal antibodies protect mice from Yersinia pestis in a bubonic plague model

Yersinia pestis is the etiologic agent of plague that has killed more than 200 million people throughout the recorded history of mankind. Antibiotics may provide little immediate relief to patients who have a high bacteremia or to patients infected with an antibiotic resistant strain of plague. Two virulent factors of Y. pestis are the capsid F1 protein and the low-calcium response (Lcr) V-protein or V-antigen that have been proven to be the targets for both active and passive immunization. There are mouse monoclonal antibodies (mAbs) against the F1- and V-antigens that can passively protect mice in a murine model of plague; however, there are no anti-Yersinia pestis monoclonal antibodies available for prophylactic or therapeutic treatment in humans. We identified one anti-F1-specific human mAb (m252) and two anti-V-specific human mAb (m253, m254) by panning a naïve phage-displayed Fab library against the F1- and V-antigens. The Fabs were converted to IgG1s and their binding and protective activities were evaluated. M252 bound weakly to peptides located at the F1 N-terminus where a protective mouse anti-F1 mAb also binds. M253 bound strongly to a V-antigen peptide indicating a linear epitope; m254 did not bind to any peptide from a panel of 53 peptides suggesting that its epitope may be conformational. M252 showed better protection than m253 and m254 against a Y, pestis challenge in a plague mouse model. A synergistic effect was observed when the three antibodies were combined. Incomplete to complete protection was achieved when m252 was given at different times post-challenge. These antibodies can be further studied to determine their potential as therapeutics or prophylactics in Y. pestis infection in humans.     

Identification and characterization of a novel agonistic anti-DR4 human monoclonal antibody

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its functional receptors, DR4 and DR5, have been established as promising targets for cancer treatment. Therapeutics targeting TRAIL and its receptors are not only effective in killing many types of tumors, but they also synergize with traditional therapies and show efficacy against tumors that are otherwise resistant to conventional treatments. We describe here the identification and characterization of two human monoclonal antibodies, m921 and m922, that are specific for human DR4. Both antibodies competed with TRAIL for binding to DR4, but only m921 recognized cell surface-associated DR4 and inhibited the growth of ST486 cells. This antibody may have potential for further development as a candidate therapeutic and research tool.Key words: DR4, TRAIL, lymphoma, therapeutic antibody     

Effects of growth factors FGF4, TGFα, and TGFβ1 on development of parthenogenetic embryos of C57BL/6 mice

We studied the effects of three growth factors, fibroblast growth factor (FGF4), transforming growth factor (TGF), and transforming growth factor 1 (TGF1), on development of diploid parthenogenetic embryos of C57BL/6 mice, which are not capable of developing to somatic stages. Parthenogenetic embryos were treated with growth factors at optimal doses in vitro at the morula-blastocyst stages and transplanted in the uterus of pseudopregnant females. FGF4 and TGF improved the development of parthenogenetic embryos at the preimplantation stages and the number of blastocysts increased under the influence of TGF. All three growth factors improved the implantation of embryos in the uterus. When FGF4 or TGF1 2.4 were added to the nutrient medium, 2.4 or 1.6%, respectively, of parthenogenetic embryos reached the somatic stages in utero. No somitic embryos were observed in the control. The treatment of parthenogenetic embryos with two growth factors, FGF4 and TGF1 , simultaneously increased the amount of somatic embryos to 7.5%, while combination of three growth factors in creased the amount of such embryos to 16.7%. In the latter case, some parthenogenetic embryos reached the stage of 25–27 pairs of somites and were 2.0–2.5 mm long. The data we obtained suggest that, when combined, the growth factors FGF4, TGF, and TGF1 possessed a synergistic effect leading to a significant improvement of the development of parthenogenetic C57BL/6 embryos.__________Translated from Ontogenez, Vol. 36, No. 2, 2005, pp. 145–150.Original Russian Text Copyright © 2005 by Penkov, Platonov, Dimitrov, Mironova, Konyukhov.