A near-infrared method for studying hydration changes in aqueous solution: Illustration with protease reactions and protein denaturation

Proteins undergoing protease reactions, heat denaturation, or interactions with sodium dodecyl sulfate (SDS) were used to demonstrate the effectiveness of a near-infrared method for the quantitative study of changes in hydration or water binding during such processes. The spectra of different proteins showed that the liberation of $\text{COO}{}^{\mathrm{?}}\text{and NH}{}_3{}^{\mathrm{+}}$ groups during a protease reaction is associated with a large increase in hydration and excluded volume. On the basis of experiments with model compounds, other spectral changes, including development of continuum absorbance between 1.55 and 1.85 μm and a band with a peak near 2.1 μm, were also attributed to the liberation of these groups. After heat denaturation or in the presence of SDS, the rate of proteolytic hydrolysis was markedly increased, consistent with the view that some preliminary denaturation is necessary for protease activity. The validity of the hydration changes calculated for protease reactions was supported by model studies with l-lysine, and with poly-l-lysine before and after hydrolysis. The near-infrared spectrum of the protein substrate with no added protease was largely unaffected by heat treatment alone, indicating that the hydration as such was not changed to a large extent by the structural modifications of denaturation. In contrast to the protease reaction, the interactions between SDS and the proteins resulted in a decrease in hydration. Results of this paper are compared with those obtained from other methods. Some unique advantages of the near-infrared method for the study of hydration changes during reactions in aqueous solution are described.     

“Unblocking” Serum Activity <Emphasis Type="Italic">in vitro</Emphasis> in the Polyoma System may Correlate with Antitumour Effects of Antiserum <Emphasis Type="Italic">in vivo</Emphasis>

In a variety of tumour systems, individuals carrying progressively growing neoplasms have lymphoid cells with a specific cytotoxic effect on cultured tumour cells from the same individual^1–4. Since the sera of tumour-bearing individuals have been shown to prevent tumour cell destruction by immune lymphocytes in vitro^2,5–8 and since this serum blocking activity appears early in primary and transplant tumour development^5,7, it has been suggested that the appearance of this serum blocking activity might be responsible for the progressive growth of tumours in individuals having cytotoxic lymphocytes. Counteraction of this blocking activity would thus be of primary importance in facilitating the function of an already existing or bolstered cell-mediated immunity. The serum blocking activity might be inhibited in various ways, by preventing the formation of blocking antibody or by interfering with its action (“unblocking”), as demonstrated in Moloney sarcoma regressor sera⁹. This type of serum also has a therapeutic effect on Moloney sarcomas in vivo^10,11, which has been tentatively attributed to its unblocking activity^8,9 or, possibly, to a complement-dependent cytotoxicity¹⁰. Tumour growth in the Moloney sarcoma system, however, might be due in part to continuous recruitment of neoplastic cells by virus-induced transformation and so the therapeutic effect could be due to a virus-neutralizing serum activity^9,10.     

On the mechanism of ATP-induced shape changes in the human erythrocyte membranes: the role of ATP

In the preceding paper (Sheetz, M. and S.J. Singer. 1977. J Cell Biol. 73:638-646) it was shown that erythrocyte ghosts undergo pronounced shape changes in the presence of mg-ATP. The biochemical effects of the action of ATP are herein examined. The biochemical effects of the action of ATP are herein examined. Phosphorylation by ATP of spectrin component 2 of the erythrocyte membrane is known to occur. We have shown that it is only membrane protein that is significantly phosphorylated under the conditions where the shape changes are produced. The extent of this phosphorylation rises with increasing ATP concentration, reaching nearly 1 mol phosphoryle group per mole of component 2 at 8mM ATP. Most of this phosphorylation appears to occur at a single site on the protein molecule, according to cyanogen bromide peptide cleavage experiments. The degree of phosphorylation of component 2 is apparently also regulated by a membrane-bound protein phosphatase. This activity can be demonstrated in erythrocyte ghosts prepared from intact cells prelabeled with [(32)P]phosphate. In addition to the phosphorylation of component 2, some phosphorylation of lipids, mainly of phosphatidylinositol, is also known to occur. The ghost shape changes are, however, shown to be correlated with the degree of phosphorylation of component 2. In such experiment, the incorporation of exogenous phosphatases into ghosts reversed the shape changes produced by ATP, or by the membrane-intercalating drug chlorpromazine. The results obtained in this and the preceding paper are consistent with the proposal that the erythrocyte membrane possesses kinase and phosphates activities which produce phosphorylation and dephosphorylation of a specific site on spectrin component 2 molecules; the steady-state level of this phosphorylation regulates the structural state of the spectrin complex on the cytoplasmic surface of the membrane, which in turn exerts an important control on the shape of the cell.     

Selective inhibition of prostaglandin biosynthesis by gold salts and phenylbutazone

Gold salts and phenylbutazone selectively inhibit the synthesis of PGF_2α and PGE₂ respectively. Lowered production of one prostaglandin species is accompanied by an increased production of the other. Selective inhibition by these drugs was observed in the presence of adrenaline, reduced glutathione and copper sulphate under conditions when most anti-inflammatory compounds inhibited PGE₂ and PGF_2α syntheses equally. It is postulated that selective inhibitors may have a different mode of action and beneficial effects may be related to the endogenous ratio of PGE to PGF required for normal function.     

Electronic aspects of enzyme catalysis proton-electron density displacements

The applicability of an electron density-proton transfer mechanism to account for the catalytic activity of enzymes has been examined in specific enzyme reactions. A concerted displacement of a number of negatively charged atomic components in one direction, countered by an associated transfer of protons or other positive components in the opposite direction appears to be involved in the reactions studied. The charges on the components, which are revealed by the Fajans electron density configurations, relate to the continuous changes of the intensities of the electrostatic interactions along the reaction co-ordinates. When the components have converged close enough together, the continuous nature of the changes corresponds to a reduction of the energy barrier due to energy gained from the bond forming process as the bond rupturing process is taking place. From this perspective, the utilization of the binding energy between the enzyme and substrate to reduce the energy barrier, the electrostatic stabilization of the transition state, and concerted, polyfunctional acid-base catalysis can all be seen as aspects of the same Coulombic process. Conformational changes may serve to extend these co-ordinated displacements over a wider area around the active site. Comparisons to other theories and supporting observations are described.     

A web server to locate periodicities in a sequence

Summary : FT is a tool written in C++, which implements the Fourier analysis method to locate periodicities in aminoacid or DNA sequences. It is provided for free public use on a WWW server with a Java interface. Availability : The server address is http://o2.db. uoa.gr/FT Contact : shamodr@atlas.uoa.gr      

The use of digital signal processors (DSPs) in real-time processing of multi-parametric bioelectronic signals.

In many cases of bioanalytical measurement, calculation of large amounts of data, analysis of complex signal waveforms or signal speed can overwhelm the performance of microcontrollers, analog electronic circuits or even PCs. One method to obtain results in real time is to apply a digital signal processor (DSP) for the analysis or processing of measurement data. In this paper we show how DSP-supported multiplying and accumulating (MAC) operations, such as time/frequency transformation, pattern recognition by correlation, convolution or filter algorithms, can optimize the processing of bioanalytical data. Discrete integral calculations are applied to the acquisition of impedance values as part of multi-parametric sensor chips, to pH monitoring using light-addressable potentiometric sensors (LAPS) and to the analysis of rapidly changing signal shapes, such as action potentials of cultured neuronal networks, as examples of DSP capability.     

The orchestration of conscious experience by subcortical structures

It is argued that conscious emotional feelings can not be adequately explained by just particular circuits or coherent activations within the brain, as is conventionally believed; nor by activations representing environmental stimuli going to the brain. According to the model suggested herein, the limbic system responds to sensory and other inputs according to how closely they are associated with built‐in rewards or punishments. It does this by (a) activating the autonomic nervous system so that it prepares the body to acquire a reward or avoid a punishment, and (b) also activating the prefrontal cortex (PFC). The PFC activations are temporally correlated with the autonomic activations and the feedback to them, so that they become identified with the autonomic attempts to acquire (a reward) or avoid (a punishment). The PFC circuit thus acquires a valence. The valence, along with arousal in a given context, underlies conscious emotional feelings. The model is related to: (a) how attention progresses along networks within working memory; (b) how a single, unified percept is formed; (c) how both value‐based and cognitive‐based responses are formulated; and (d) how the stream of consciousness is put together and driven forward. These concepts are integrated into a scenario of the orchestration of conscious experience and behaviour by subcortical‐limbic system structures interacting with the cortex, and are shown to be consistent with much of the literature.     

WFDC1/ps20 is a novel innate immunomodulatory signature protein of human immunodeficiency virus (HIV)-permissive CD4+ CD45RO+ memory T cells that promotes infection by upregulating CD54 integrin expression and is elevated in HIV type 1 infection

Understanding why human immunodeficiency virus (HIV) preferentially infects some CD4⁺ CD45RO⁺ memory T cells has implications for antiviral immunity and pathogenesis. We report that differential expression of a novel secreted factor, ps20, previously implicated in tissue remodeling, may underlie why some CD4 T cells are preferentially targeted. We show that (i) there is a significant positive correlation between endogenous ps20 mRNA in diverse CD4 T-cell populations and in vitro infection, (ii) a ps20⁺ permissive cell can be made less permissive by antibody blockade- or small-interference RNA-mediated knockdown of endogenous ps20, and (iii) conversely, a ps20^low cell can be more permissive by adding ps20 exogenously or engineering stable ps20 expression by retroviral transduction. ps20 expression is normally detectable in CD4 T cells after in vitro activation and interleukin-2 expansion, and such oligoclonal populations comprise ps20^positive and ps20^low/negative isogenic clones at an early differentiation stage (CD45RO⁺/CD25⁺/CD28⁺/CD57⁻). This pattern is altered in chronic HIV infection, where ex vivo CD4⁺ CD45RO⁺ T cells express elevated ps20. ps20 promoted HIV entry via fusion and augmented CD54 integrin expression; both of these effects were reversed by anti-ps20 antibody. We therefore propose ps20 to be a novel signature of HIV-permissive CD4 T cells that promotes infection in an autocrine and paracrine manner and that HIV has coopted a fundamental role of ps20 in promoting cell adhesion for its benefit. Disrupting the ps20 pathway may therefore provide a novel anti-HIV strategy.