Identification of trimeric peptides that bind porcine parvovirus from mixtures containing human blood plasma

Virus contamination in human therapeutics is of growing concern as more therapeutic products from animal or human sources come into the market. All biopharmaceutical processes are required to have at least two distinct viral clearance steps to remove viruses. Most of these steps work well for enveloped viruses and large viruses, whether enveloped or not. That leaves a class of small non-enveloped viruses, like parvoviruses and hepatitis A, which are not easily removed by these typical steps. In this study, we report the identification of trimeric peptides that bind specifically to porcine parvovirus (PPV) and their potential use to remove this virus from process solutions. All of the trimeric peptides isolated completely removed all detectable PPV from buffer in the first nine column volumes, corresponding to a clearance of 4.5-5.5 log of infectious virus. When the virus was spiked into a more complex matrix consisting of 7.5% human blood plasma, one of the trimers, WRW, was able to remove all detectable PPV in the first three column volumes, after which human blood plasma began to interfere with the binding of the virus to the peptide resin. These trimer resins removed considerably more virus than weak ion exchange resins. The results of this work indicate that small peptide ligand resins have the potential to be used in virus removal processes where removal of contaminating virus is necessary to ensure product safety.     

The structure of B-helical C-G-A-T-C-G-A-T-C-G and comparison with C-C-A-A-C-G-T-T-G-G. The effect of base pair reversals

The crystal structure of the DNA decamer C-G-A-T-C-G-A-T-C-G has been solved to a resolution of 1.5 A, with a final R-factor of 16.1% for 5,107 two-sigma reflections. Crystals are orthorhombic space group P2(1)2(1)2(1), with cell dimensions a = 38.93 A, b = 39.63 A, c = 33.30 A, and 10 base pairs/asymmetric unit. The final structure contains 404 DNA atoms, 142 water molecules treated as oxygen atoms, and two Mg(H2O)6(2+) complexes. Decamers stack atop one another to simulate continuous helical columns through the crystal, as with three previously solved monoclinic decamers, but the lateral contacts between columns are quite different in the orthorhombic and monoclinic cells. Narrow and wide regions of the minor groove exhibit a single spine or two ribbons of hydration, respectively, and the minor groove is widest when BII phosphate conformations are opposed diagonally across the groove. Phosphate conformation, in turn, appears to have a base sequence dependence. Twist, rise, cup, and roll are linked as has been observed in the three monoclinic decamers and can be characterized by high or low twist profiles. In all five known decamer crystal structures and eight representative dodecamers, a high twist profile is observed with G-C and G-A steps whereas all other R-R steps are low twist profiles (R = purine). A-T and A-C steps are intermediate in character whereas C-A and C-G exhibit behavior that is strongly influenced by the profiles of the preceding and following steps. When sufficient data are in hand, sequence/structure relationships for all helix parameters probably should be considered in a 4-base pair context. At this stage of limited information the problem is compounded because there are 136 unique 4-base steps x-A-B-y in a double helix as compared with only 10 2-base steps A-B.     

Discontinuous release of heat at successive steps of oxygenation in human and bovine hemoglobin at pH 9.0

We have measured the temperature dependence of the oxygen-binding isotherms of human and bovine hemoglobin at pH 9.0 in 0.1 M borate buffer. In both hemoglobins the ionization of the Bohr protons is finished at this pH; therefore, their heat does not interfere with the measurements. Two sets of curves have been obtained, which have been analyzed by either singular or global procedures for estimating the enthalpy changes of subsequent steps of oxygenation. The data indicate that in human hemoglobin the reaction with oxygen is enthalpy driven for steps 1, 2, and 4 while it is entropy driven for step 3. In bovine hemoglobin this phenomenon is even more evident: steps 2 and 4 are enthalpy driven while steps 1 and 3 are entropy driven. The discontinuous distribution of heat at subsequent steps of oxygenation suggests that the T to R transition in hemoglobin is not a monotonic process and involves conformations with novel characteristics.     

Measuring kinesin's first step

A variety of models have recently emerged to explain how the molecular motor kinesin is able to maintain processive movement for over 100 steps. Although these models differ in significant features, they all predict that kinesin's catalytic domains intermittently separate from each other as the motor takes 8-nm steps along the microtubule. Furthermore, at some point in this process, one molecule of ATP is hydrolyzed per step. However, exactly when hydrolysis and product release occur in relation to this forward step have not been established. Furthermore, the rate at which this separation occurs as well as the speed of motor stepping onto and release from the microtubule have not been measured. In the absence of this information, it is difficult to critically evaluate competing models of kinesin function. We have addressed this issue by developing spectroscopic probes whose fluorescence is sensitive to motor-motor separation or microtubule binding. The kinetics of these fluorescence changes allow us to directly measure how fast kinesin steps onto and releases from the microtubule and provide insight into how processive movement is maintained by this motor.     

Effects of cell cycle status on early events in retroviral replication

The study of retroviruses over the last century has revealed a wide variety of disease-producing mechanisms, as well as apparently harmless interactions with animal hosts. Despite their potential pathogenic properties, the intrinsic features of retroviruses have been harnessed to create gene transfer vectors that may be useful for the treatment of disease. Retroviruses, as all viruses, have evolved to infect specific cells within the host, and such specificities are relevant to both pathogenesis and retrovirus-based vector design. The majority of cells of an animal host are not progressing rapidly through the cell cycle, and such a cellular environment appears to be suboptimal for replication of all retroviruses. Retrovirus-based vectors can therefore be restricted in many important target cells, such as post-mitotic differentiated cells or stem cells that may divide only infrequently. Despite intense interest, our understanding of how cell cycle status influences retroviral infection is still quite limited. In this review, we focus on the importance of the cell cycle as it relates to the early steps in retroviral replication. Retroviruses have been categorized based on their abilities to complete these early steps in non-cycling cells. However, all retroviruses are subject to a variety of cell cycle restrictions. Here, we discuss such restrictions, and how they may block retroviral replication, be tolerated, or overcome.     

On the origin and evolution of the genetic code I. Wobbling and its potential significance

In this paper several properties of the genetic code are interpreted by assuming that wobbling or some remnant of wobbling has originally been a common phenomenon also in the first nucleotide of each codon, and not only in the third nucleotide. Some of the last steps in the evolution of the genetic code are described on the basis of this interpretation of genetic code features.An attempt to outline some of the earlier steps in the evolution of the genetic code is based on the assumption that at an earlier stage wobbling may also have been common in the central nucleotide of each codon.In the last part of the paper the possibility is considered that the pairing rules which characterize wobbling may have been much more common in the past not only in codon-anticodon pairing but also in polymer copying. The advantages of a freer purine-pyrimidine pairing like the one characteristic of wobbling in a primitive (or prebiologic) environment in which nucleotide production was not entirely (or not at all) under biologic control are stressed.This paper is based exclusively on the “Frozen accident” interpretation of the genetic code (Crick, 1968) with a few modifications introduced or implied in the text. No stereochemical codon interpretations and only a minimum of chemical considerations are involved.     

The effects of sequence context on base dynamics at TpA steps in DNA studied by NMR.

Base dynamics, heretofore observed only at TpA steps in DNA, were investigated as a function of sequence context by NMR spectroscopy. The large amplitude conformational dynamics have been previously observed in TnAn segments where n > or = 2. In order to determine whether the dynamic characteristics occur in more general sequence contexts, we examined four self-complementary DNA sequences, [d(CTTTA-NATNTAAAG)2] (where N = A, C, T, G and N = complement of N). The anomalous broadening of the TpA adenine H2 resonance which is indicative of large amplitude base motion was observed in all nine unique four nucleotide contexts. Furthermore, all the adenine H2 resonances experienced a linewidth maximum as a function of temperature, which is a characteristic of the dynamic process. Interestingly, the temperature of the linewidth maximum varied with sequence indicating that the thermodynamics of TpA base dynamics are also sequence dependent. In one example, neither a T preceding nor an A trailing the TpA step was required for base dynamics. These results show that base dynamics, heretofore observed in only a few isolated sequences, occurs at all TpA steps which are either preceded or followed by a thymine or adenine, respectively, and may be characteristic of all TpA steps in DNA notwithstanding sequence context.     

Invariant and variable base stacking geometries in B-DNA and A-DNA

We calculated the interatomic distances between all couples of non-hydrogen atoms belonging to the neighboring Watson-Crick base pairs in the available crystal structures of DNA. Their standard deviations revealed remarkably large differences in the variability of the base stacking geometries of the particular steps. In line with experimental studies in solution, (CpA)-(TpG) and (TpA).(TpA) were identified as the most variable or flexible steps in the crystal structures of B-DNA. On the other hand, base stacking geometries of the (ApT).(ApT) steps were the most invariant, which was very surprising because all three steps composed only of C and G were much more flexible. This finding suggests that conformational stability of DNA and the rigidity have different origins. Furthermore, the nucleotide sequence dependence of the flexibility was almost reversed in A-DNA because the most flexible steps in B-DNA were the least flexible in A-DNA. The most invariant steps of B-DNA were variable in A-DNA. The (ApT).(ApT) step was a notable exception to this rule because it belonged to the most rigid steps in both B-DNA and A-DNA. The present results are fully consistent with the properties that poly(dA-dT).poly(dA-dT), poly(dA).poly(dT), poly(dAdC).poly(dG-dT) and poly(dA-dG).poly(dC-dT) exhibit in solution.     

Electrochemical Studies of Tirapazamine: Generation of the One-Electron Reduction Product

The electrochemical properties of the benzotriazine di-N-oxide, tirapazamine (SR4233), and the mono-and zero-N-oxides, SR4317 and SR4330 respectively, have been investigated in dimethylformamide and acetonitrile. The voltammetry of tirapazamine is complicated, with up to 6 reduction steps being identified, depending on the solvent. Both SR4317 and SR4330 show two reduction steps. The first reduction of all three compounds is a reversible or quasi-reversible step, which is assigned to a 1-electron addition. Cyclic voltammetric studies show that the anion radical product is stable, although the tirapazamine 1-electron addition product shows a tendency to participate in a chemical following reaction. Subsequent reduction steps are all highly irreversible in nature. The 2nd electron transfer of SR4317 results in the formation of the free base, SR4330, which is identified voltammetrically. Comparison is made with the voltammetric behaviour of quinoline and quinoline-oxide.     

Vesicle docking in regulated exocytosis

In electron micrographs, many secretory and synaptic vesicles are found 'docked' at the target membrane, but it is unclear why and how. It is generally assumed that docking is a necessary first step in the secretory pathway before vesicles can acquire fusion competence (through 'priming'), but recent studies challenge this. New biophysical methods have become available to detect how vesicles are tethered at the target membrane, and genetic manipulations have implicated many genes in tethering, docking and priming. However, these studies have not yet led to consistent working models for these steps. In this study, we review recent attempts to characterize these early steps and the cellular factors to orchestrate them. We discuss whether assays for docking, tethering and priming report on the same phenomena and whether all vesicles necessarily follow the same linear docking–priming–fusion pathway. We conclude that most evidence to date is consistent with such a linear pathway assuming several refinements that imply that some vesicles can be nonfunctionally docked ('dead-end' docking) or, conversely, that the linear pathway can be greatly accelerated (crash fusion).     

The nicking step in V(D)J recombination is independent of synapsis: implications for the immune repertoire

In all of the transposition reactions that have been characterized thus far, synapsis of two transposon ends is required before any catalytic steps (strand nicking or strand transfer) occur. In V(D)J recombination, there have been inconclusive data concerning the role of synapsis in nicking. Synapsis between two 12-substrates or between two 23-substrates has not been ruled out in any studies thus far. Here we provide the first direct tests of this issue. We find that immobilization of signals does not affect their nicking, even though hairpinning is affected in a manner reflecting its known synaptic requirement. We also find that nicking is kinetically a unireactant enzyme-catalyzed reaction. Time courses are no different between nicking seen for a 12-substrate alone and a reaction involving both a 12- and a 23-substrate. Hence, synapsis is neither a requirement nor an effector of the rate of nicking. These results establish V(D)J recombination as the first example of a DNA transposition-type reaction in which catalytic steps begin prior to synapsis, and the results have direct implications for the order of the steps in V(D)J recombination, for the contribution of V(D)J recombination nicks to genomic instability, and for the diversification of the immune repertoire.     

Information transfer in metabolic pathways. Effects of irreversible steps in computer models.

Various metabolic models have been studied by computer simulation in an effort to understand why allowing for the reversibility of the reaction catalysed by pyruvate kinase, normally considered as irreversible for all practical purposes, significantly altered the behaviour of the model of glycolysis in Trypanosoma brucei [Eisenthal, R. & Cornish-Bowden, A. (1998) J. Biol. Chem. 273, 5500-5505]. Studies of several much simpler models indicate that the enzymes catalysing early steps in a pathway must receive information about the concentrations of the metabolites at the end of the pathway if a model is to be able to reach a steady state; treating all internal steps as reversible is just one way of ensuring this. Feedback inhibition provides a much better way, and as long as feedback loops are present in a model it makes almost no difference to the behaviour whether the intermediate steps with large equilibrium constants are treated as irreversible. In the absence of feedback loops, ordinary product inhibition of all the enzymes in the chain can also transfer information; this is efficient for regulating fluxes but very inefficient for regulating intermediate concentrations. More complicated patterns of regulation, such as activation of a competing branch or forcing flux through a parallel route, can also serve to some degree as ways of passing information around an irreversible step. However, they normally do so less efficiently than inhibition, because the extent to which an enzyme or a pathway can be activated always has an upper limit (which may be below what is required), whereas most enzymes are inhibited completely at saturating concentrations of inhibitor.     

Stock enhancement as a fisheries management tool

Stock enhancement has been viewed as a positive fisheries management tool for over 100 years. However, decisions to undertake such activities in the past have often been technology-based, i.e., driven by the ability to produce fishes, with most stock enhancement projects having limited or no demonstrated success. The reasons for this have been due to an inability to identify and/or control the underlying reasons why a fishery is under-performing or not meeting management objectives. Further, stock enhancement has often been applied in isolation from other fisheries management tools (e.g., effort control). To address these issues and consider stock enhancement in a broader ecosystem perspective, a new approach for stock enhancement is proposed. The proposed model comprises four major steps; a review of all information about an ecosystem/fishery/stock and the setting of clear management targets; a comparison of all relevant fisheries management tools with the potential to meet the management targets; the instigation of a scientifically based, pilot-scale, stock enhancement program with clear objectives, targets, and evaluations; and a full-scale stock enhancement program if the pilot project meets the objectives. The model uses a flow-chart that highlights a broad range of scientific and other information, and the decisions that need to be made in relation to stock enhancement and fisheries management in general. In this way all steps are transparent and all stakeholders (managers, scientists, extractive and non-extractive users, and the general public) can contribute to the information collection and decision making processes. If stock enhancement is subsequently identified as the most-appropriate tool, then the stepwise progression will provide the best possible chance of a positive outcome for a stock enhancement project, while minimizing risks and costs. In this way, stock enhancement may advance as a science and develop as a useful fisheries management tool in appropriate situations.     

Effects of the protein matrix on glycan processing in glycoproteins

In the biosynthesis of glycoproteins containing asparagine-linked glycans, a number of regulatory factors must be involved in converting the single glycan precursor into the variety of different final structures observed in different eukaryotic species. Among these factors are the kind of glycan-processing enzymes available in the Golgi apparatus of different cells, the specificity and regulatory properties of these enzymes, and the unique properties of the protein matrix in which a given glycan resides during the biosynthetic processing. In examining the role of this latter regulatory factor, we have considered a simplified model in which a few key steps are common to all cells, regardless of the nature of the processing enzymes available. The protein-bound oligomannose precursor Man8GlcNAc2-, arriving in the Golgi after the initial trimming in the endoplasmic reticulum (ER), first undergoes a series of preprocessing steps to yield Man5GlcNAc2- in animals and plants or Man13-15GlcNAc2- in yeast. At this stage the key commitment step--to process or not to process--determines whether the above intermediates will remain as unprocessed oligomannose structures or be initiated into a new series of reactions to yield processed structures characteristic of the organisms involved (complex or hybrid for vertebrates, polymannose for yeast, xylosylated glycans for plants and some invertebrates, or Man3GlcNAc2- structures for other invertebrates). It is proposed that this commitment step, along with the obligatory preprocessing steps, is regulated primarily by each glycan's unique exposure on its protein matrix. Subsequent processing steps leading to complex or hybrid structures, fucosylation, extent of branching, and specific structures at the nonreducing terminals are most likely determined primarily by the enzyme makeup of the individual processing machineries, but with the protein matrix still playing a significant role.     

Dynamical properties of min-max networks

In this paper we study the dynamical behavior of a class of neural networks where the local transition rules are max or min functions. We prove that sequential updates define dynamics which reach the equilibrium in O(n2) steps, where n is the size of the network. For synchronous updates the equilibrium is reached in O(n) steps. It is shown that the number of fixed points of the sequential update is at most n. Moreover, given a set of p < or = n vectors, we show how to build a network of size n such that all these vectors are fixed points.     

Witch's chin: a progressive, three-step technique

Witch's chin is an unpleasant aesthetic defect characterized by ptosis of premental tissue and a deep submental fold, which may be exaggerated by hyperprojection of the mandible. These three elements determine the different degrees of deformity; therefore, the ideal treatment should be directed to one, two, or all three of them. Despite unanimity on the surgical approach of the defect, a large variety of techniques have been proposed by various authors. The need to use a technique suitable for different clinical pictures, characterized by a progressive surgical aggression, as usually performed in this practice, has led to standardize a technique to correct witch's chin, by means of three progressive steps, depending on the degree of deformity. The advantage of this procedure is that once a good result has been achieved, the subsequent steps may be omitted. The technique has been successfully performed in five patients, and the mean follow-up is 12 months. Figures from two representative cases are presented.     

Methanogenic archaea: ecologically relevant differences in energy conservation

Most methanogenic archaea can reduce CO(2) with H(2) to methane, and it is generally assumed that the reactions and mechanisms of energy conservation that are involved are largely the same in all methanogens. However, this does not take into account the fact that methanogens with cytochromes have considerably higher growth yields and threshold concentrations for H(2) than methanogens without cytochromes. These and other differences can be explained by the proposal outlined in this Review that in methanogens with cytochromes, the first and last steps in methanogenesis from CO(2) are coupled chemiosmotically, whereas in methanogens without cytochromes, these steps are energetically coupled by a cytoplasmic enzyme complex that mediates flavin-based electron bifurcation.