The HIV-1 gp41 N-terminal heptad repeat plays an essential role in membrane fusion

For many different enveloped viruses the crystal structure of the fusion protein core has been established. A striking conservation in the tertiary and quaternary arrangement of these core structures is repeatedly revealed among members of diverse families. It has been proposed that the primary role of the core involves structural rearrangements which facilitate apposition between viral and target cell membranes. Forming the internal trimeric coiled coil of the core, the N-terminal heptad repeat (NHR) of HIV-1 gp41 was suggested to have additional roles, due to its ability to bind biological membranes. The NHR is adjacent to the N-terminal hydrophobic fusion peptide (FP), which alone can fuse biological membranes. To investigate the role of the NHR in membrane fusion, we synthesized and functionally characterized HIV-1 gp41 peptides corresponding to the FP and NHR alone, as well as continuous peptides made of both FP and NHR (wild type and mutant). We show here that a consecutive, 70-residue peptide consisting of both the FP and NHR (gp41/1-70) has dramatic fusogenic properties. The effect of including the complete NHR, as compared to shorter 23-, 33-, or 52-residue N-terminal peptides, is illustrated by a leap in lipid mixing of phosphatidylcholine (PC) large unilamellar vesicles (LUV) and clearly delineates the synergistic role of the NHR in the fusion event. Furthermore, a mutation in the NHR that renders the virus noninfectious is reflected by a significant reduction in in vitro lipid mixing induced by the mutant, gp41/1-70 (I62D). Additional spectroscopic studies, characterizing membrane binding and apposition induced by the peptides, help to clarify the role of the NHR in membrane fusion.     

Effect of flow regime on the architecture of a Pseudomonas fluorescens biofilm

A comparison of the effects of laminar versus turbulent flow regime on the characteristics of a single-species biofilm is presented. The study was carried out by growing Pseudomonas fluorescens biofilms in a flow cell and studying the different layers of the biological matrix with a confocal laser-scanning microscope. The following conclusions were obtained: i) a higher concentration of cells was found in the upper layers of the microbial films than in their inner layers, regardless of the flow regime; ii) the fraction of cells in the overall biofilm mass decreased with time as the film grew; and iii) under laminar flow the total number of cells was higher than in biofilms formed under turbulent flow, but the latter had a higher number of cells per unit volume. Such conclusions, together with the fact that the biofilms were more dense and stable when formed in contact with turbulent flows, favor the design of more compact and efficient biofilm reactors operating in turbulent conditions.     

Experience sampling methodology in mental health research: new insights and technical developments

In the mental health field, there is a growing awareness that the study of psychiatric symptoms in the context of everyday life, using experience sampling methodology (ESM), may provide a powerful and necessary addition to more conventional research approaches. ESM, a structured self‐report diary technique, allows the investigation of experiences within, and in interaction with, the real‐world context. This paper provides an overview of how zooming in on the micro‐level of experience and behaviour using ESM adds new insights and additional perspectives to standard approaches. More specifically, it discusses how ESM: a) contributes to a deeper understanding of psychopathological phenomena, b) allows to capture variability over time, c) aids in identifying internal and situational determinants of variability in symptomatology, and d) enables a thorough investigation of the interaction between the person and his/her environment and of real‐life social interactions. Next to improving assessment of psychopathology and its underlying mechanisms, ESM contributes to advancing and changing clinical practice by allowing a more fine‐grained evaluation of treatment effects as well as by providing the opportunity for extending treatment beyond the clinical setting into real life with the development of ecological momentary interventions. Furthermore, this paper provides an overview of the technical details of setting up an ESM study in terms of design, questionnaire development and statistical approaches. Overall, although a number of considerations and challenges remain, ESM offers one of the best opportunities for personalized medicine in psychiatry, from both a research and a clinical perspective.     

Social stimuli as incentives for delayed response performance by infant pigtailed macaques (Macaca nemestrina)

Six pigtailed macaque monkeys (Macaca nemestrina), ranging in age from 4.5 to 13.5 months, were studied longitudinally on a delayed response procedure in a spatial choice apparatus. In each trial the subjects were exposed to two stimulus animals, an unfamiliar adult female and a familiar age-mate. Prior work showed that such nursery-reared infants overwhelmingly prefer a familiar peer over an unfamiliar adult female. Therefore, the peer was considered to be a positive incentive and choosing the peer was defined as the “correct” response. After the infants were given visual exposure to the stimulus animals, an opaque door was lowered to block them from the subject's view. Then a single delay period of 0, 5, 15, 60, 120, 240, or 480 sec was introduced during 20-trial sessions. The delays were increased over sessions, with about two weeks between sessions. The subjects reliably chose the familiar peer after delays of up to 60 sec, with one subject maintaining correct choices even after 8-min delays. These results revealed that the delayed response performance of young macaques with social incentives was as good as, or better than, maximum performance levels reported for macaques with food incentives. Cues such as odors, sounds, and visual-postural orientation sets could not explain the performance levels at long delays found in this study.     

CARBONIC ANHYDRASE IN THE CHLOROPLAST OF A COCCOLITHOPHORID (PRYMNESIOPHYCEAE)1

The activity and subcellular distribution of carbonic anhydrase in a coccolithophorid alga, CCMP 299, was examined. The enzyme could not be detected in crude cell homogenates but was present at high specific activity (27.5 unit·mg^?1 protein) in chloroplasts (density, 1.14 g·cm^?3) isolated in a sucrose gradient. The carbonic anhydrase activity was sensitive to known inhibitors. Inhibition at 50% (I₅₀) was obtained with concentrations of 4.60 mM and 2.65 mM for acetazolamide and NaN₃, respectively. These levels are more consistent with patterns of inhibition previously observed for chloroplastic (as compared to periplasmic) carbonic anhydrase. In this organism, carbonic anhydrase was localized in the chloroplast stroma. These findings are discussed in terms of the relationship among dissolved inorganic carbon interconversions, photosynthesis, and calcification.     

Thermodynamics of reversible monomer-dimer association of tubulin

The equilibrium between the rat brain tubulin alpha beta dimer and the dissociated alpha and beta monomers has been studied by analytical ultracentrifugation with use of a new method employing short solution columns, allowing rapid equilibration and hence short runs, minimizing tubulin decay. Simultaneous analysis of the equilibrium concentration distributions of three different initial concentrations of tubulin provides clear evidence of a single equilibrium characterized by an association constant, Ka, of 4.9 X 10(6) M-1 (Kd = 2 X 10(-7) M) at 5 degrees, corresponding to a standard free energy change on association delta G degrees = -8.5 kcal mol-1. Colchicine and GDP both stabilize the dimer against dissociation, increasing the Ka values (at 4.5 degrees C) to 20 X 10(6) and 16 X 10(6) M-1, respectively. Temperature dependence of association was examined with multiple three-concentration runs at temperatures from 2 to 30 degrees C. The van't Hoff plot was linear, yielding positive values for the enthalpy and entropy changes on association, delta S degrees = 38.1 +/- 2.4 cal deg-1 mol-1 and delta H degrees = 2.1 +/- 0.7 kcal mol-1, and a small or zero value for the heat capacity change on association, delta C p degrees. The entropically driven association of tubulin monomers is discussed in terms of the suggested importance of hydrophobic interactions to the stability of the monomer association and is compared to the thermodynamics of dimer polymerization.     

Biotinylated endothelin as a probe for the endothelin receptor

Biotinylated derivatives of endothelin (ET)-1 were prepared by chemical modification of ET-1 with sulfosuccinimidyl 6-(biotinamido) hexanoate. Two major biotinylated ET analogs were purified by reversed-phase high performance liquid chromatography. Edman degradation indicated that the first eluting peptide contains one biotin residue on lysine at position 9, while the second derivative contains an additional biotin residue at position 1. Competition binding studies to mouse osteoblastic cell line MC3T3-E1 using ¹²⁵I-labeled ET-1 revealed IC₅₀ values of 5, 30 and 600 nM for native ET, the mono- and the dibiotinylated ET analog, respectively. A similar order of potency was obtained when these ET derivatives were examined for stimulation of DNA synthesis in MC3T3-E1 cells. In addition, incubation of MC3T3-E1 cells with the monobiotinylated ET and subsequent addition of rhodamine-avidin resulted in an evenly distributed fluorescence over the cell surface. The fluorescence observed was completely abolished in the presence of an excess of native ET. Thus the monobiotinylated ET proves to be useful for localization of the ET receptors.     

Correlation between complementation group for immortality and DNA synthesis inhibitors

Previous studies had demonstrated that a DNA synthesis inhibitor(s) was produced by senescent but not young human diploid fibroblasts (HDF). Analysis of immortal human cell lines led to the finding that SUSM-1, carcinogen-treated immortal human liver fibroblast cells, expressed a potent inhibitor of DNA synthesis that was active in proliferation-competent young HDF but did not affect the SUSM-1 cell line itself. To determine whether one mechanism of escape from senescence to the immortal phenotype involved the loss of response to such DNA synthesis inhibitors, we initiated the present study analyzing a larger number of immortal human cell lines representative of the four complementation groups for indefinite division identified to date. We have found a correlation between the assignment of a cell line to Complementation Group D and the production of DNA synthesis inhibitors coupled with inability to respond to the inhibitory factors. We have also observed a correlation between the ability of immortal cell lines to respond to such DNA synthesis inhibitory factors and assignment to Complementation Group B. These data suggest DNA synthesis inhibitors are involved in the limited lifespan of normal cells and that the immortalization process may involve alterations in the activity of or response to such inhibitors.     

Two forms of DNA polymerase delta from mouse cells. Purification and properties

A procedure is described for the purification from cultured mouse cells of two DNA polymerase "delta-like" enzymes, as defined by intrinsic 3'-exonuclease activity, inhibition by aphidicolin, and relative insensitivity to N2-(p-n-butylphenyl)-dGTP. One of the two enzymes has been purified to near homogeneity and, similar to the DNA polymerase delta from calf thymus described by Lee et al. (Lee, M. Y. W. T., Tan, C. K., Downey, K. M., and So, A. G. (1984) Biochemistry 23, 1906-1913), it has a total molecular mass of 178 kDa (from sedimentation velocity of 8.0 S and Stokes radius of 54 A) and is composed of one each of 125- and 50-kDa polypeptides. It also resembles the DNA polymerase delta of Lee et al. in being stimulated by proliferating cell nuclear antigen (PCNA). It is the first clear structural and functional counterpart of the calf thymus enzyme. The major difference between the mouse DNA polymerase delta and the calf thymus enzyme of Lee et al. is that, under specific conditions, the mouse enzyme is active with poly(dA).oligo(dT) in the absence of PCNA, whereas the activity of the calf thymus enzyme with this template is reported to be completely dependent on PCNA. The reason for this difference is not known at this time. The second mouse cell enzyme has a molecular mass of 112 kDa (from sedimentation velocity of 6.3 S and Stokes radius of 43.0 A) and consists of a single polypeptide of 123-125 kDa in denaturing gels (p125). On the basis of its apparent formation by dissociation of DNA polymerase delta, and multiple similarities with DNA polymerase delta in enzymatic properties, the p125 is provisionally identified as the 125-kDa polypeptide of DNA polymerase delta. The p125 does not respond to PCNA, suggesting that the 50-kDa polypeptide is required for the stimulation of DNA polymerase delta by PCNA. The presence of the p125 in cell extracts would explain reports that DNA polymerase delta consists of a single polypeptide of approximately 125 kDa and/or thast it has a smaller molecular mass than DNA polymerase delta of Lee et al. and is not affected by PCNA (this does not apply to PCNA-independent DNA polymerase delta-like enzymes with higher molecular mass than the polymerase delta of Lee et al., which have recently been named DNA polymerases epsilon).