Binding of alkyl polyglucoside surfactants to bacteriorhodopsin and its relation to protein stability

The binding of alkyl polyglucoside surfactants to the integral membrane protein bacteriorhodopsin (BR) and the formation of protein-surfactant complexes are investigated by sedimentation equilibrium via analytical ultracentrifugation and by small-angle neutron scattering (SANS). Contrast variation techniques in SANS enable measurement of the composition of the protein-surfactant complexes and determination of the thickness of the surfactant shell bound to the protein. The results indicate that alkyl polyglucosides can bind to BR as single surfactant layers or as a thicker shell. The thickness of the surfactant shell increases with increasing surfactant tail length, and it is generally unrelated to the aggregation number of the micelles even for a small and predominantly hydrophobic membrane protein such as BR. The aggregation numbers determined by sedimentation equilibrium methods match those measured by SANS, which also allows reconstruction of the shape of the protein-detergent complex. When the surfactant is present as a single layer, the BR loses activity, as measured by absorption spectroscopy, more quickly than it does when the surfactant forms a thicker shell.     

How to (or not to) communicate science

Protagonists for 'the public understanding of science' still sometimes fail to recognize that there is also a need for 'the scientists' understanding of the public' and that for most of science most of the time we are all public. 'Science' is communicated to 'the public' through popular books, museums, TV, the Internet, but far too often the present state of scientific belief is presented uncritically as the onward march of truth as discovered by Euro-American males. This has contributed to a widespread public concern, if not mistrust, in many areas of science, not least genetics and neuroscience. Although researchers often criticize the media for misrepresenting their work, the hype and simplifications often begin with the press releases put out by the researchers, their institutions and the scientific journals themselves. I conclude by looking more optimistically at the ways in which, by bringing natural science into theatre, novels and other art forms, the fragmentation of our culture may be diminished.     

Industry's challenge to academia: changing the bench to bedside paradigm

The need for interdisciplinary collaboration is arising as a result of accelerating advances in basic science, including massive research and development funding by both government and industry, which has spurred the so-called "nanotechnology revolution" and developments at the intersection of the life and physical sciences, increasing emphasis by federal research funding agencies on interdisciplinary and inter-institutional research and by market influences. A number of barriers presently limit the interaction between academics and industry, including the typically very time-consuming and slow pace of technology transfer, which is compounded in the case of interdisciplinary and inter-institutional licensing, as well as the natural, and understandable, antipathies that exist between academia and industry as a result of their differing missions and approaches to scientific discovery. Moreover, if mechanisms are not in place at the outset of an inter-university collaboration, then the transition of inventions to clinical applications can be fraught with additional complexities and barriers. Policies suggested by the National Nanotechnology Initiative offer a number of ideas for overcoming barriers to multidisciplinary and inter-institutional research and illustrate some of the ways in which academia can structure partnerships with industry that will not only provide needed funding for multidisciplinary and inter-institutional biomedical research in an era of diminishing federal resources, but may permit academia, on the one hand, and industry, on the other, to benefit from the strengths provided by the other without compromising either academia's or industry's basic missions.     

Myosin-Va binds to and mechanochemically couples microtubules to actin filaments

Myosin-Va was identified as a microtubule binding protein by cosedimentation analysis in the presence of microtubules. Native myosin-Va purified from chick brain, as well as the expressed globular tail domain of this myosin, but not head domain bound to microtubule-associated protein-free microtubules. Binding of myosin-Va to microtubules was saturable and of moderately high affinity (approximately 1:24 Myosin-Va:tubulin; Kd = 70 nM). Myosin-Va may bind to microtubules via its tail domain because microtubule-bound myosin-Va retained the ability to bind actin filaments resulting in the formation of cross-linked gels of microtubules and actin, as assessed by fluorescence and electron microscopy. In low Ca2+, ATP addition induced dissolution of these gels, but not release of myosin-Va from MTs. However, in 10 microM Ca2+, ATP addition resulted in the contraction of the gels into aster-like arrays. These results demonstrate that myosin-Va is a microtubule binding protein that cross-links and mechanochemically couples microtubules to actin filaments.     

Ripped to death

An old puzzle in the field of cell death was solved recently: the mysterious embryonic lethality of animals deficient in caspase-8 or Fas-associated death domain (FADD), proteins involved in a pathway of apoptosis. This lethality is caused by a failure to develop the yolk sac vasculature rather than a lack of apoptosis. Remarkably, development is rescued by ablation of either of two receptor interacting serine-threonine kinases (RIPKs). Despite being well known cell killers, caspase-8 and FADD act together to block RIPK-mediated necrosis. To manifest this newly elucidated pro-survival function, FADD and caspase-8 depend on FLIP(Long), a catalytically inactive caspase-8 homolog. In this review, the mechanism by which RIPK necrotic death is inhibited by this trio is discussed, as well as how RIPKs might themselves mediate cell death.     

Many roads to resistance: how invertebrates adapt to Bt toxins

The Cry family of Bacillus thuringiensis insecticidal and nematicidal proteins constitutes a valuable source of environmentally benign compounds for the control of insect pests and disease agents. An understanding of Cry toxin resistance at a molecular level will be critical to the long-term utility of this technology; it may also shed light on basic mechanisms used by other bacterial toxins that target specific organisms or cell types. Selection and cross-resistance studies have confirmed that genetic adaptation can elicit varying patterns of Cry toxin resistance, which has been associated with deficient protoxin activation by host proteases, and defective Cry toxin-binding cell surface molecules, such as cadherins, aminopeptidases and glycolipids. Recent work also suggests Cry toxin resistance may be induced in invertebrates as an active immune response. The use of model invertebrates, such as Caenorhabditis elegans and Drosophila melanogaster, as well as advances in insect genomics, are likely to accelerate efforts to clone Cry toxin resistance genes and come to a detailed and broad understanding of Cry toxin resistance.     

A refined technique to apply electrical currents to callus cultures

A simple, inexpensive technique is described which can be used to pass small electrical currents of a few microamperes through callus cultures under sterile conditions without contaminating the tissue or the medium with electrode products. Application of 1 or 2 microamperes currents of either polarity stimulated growth as well as shoot regeneration in tobacco (Nicotiana tabacum var. virginica) callus cultures, indicating that these effects are due to the electrical fields set up by the exogenous currents, rather than by electrode products.     

The binding cascade of SecB to SecA to SecY/E mediates preprotein targeting to the E. coli plasma membrane

The export of many E. coli proteins such as proOmpA requires the cytosolic chaperone SecB and the membrane-bound preprotein translocase. Translocase is a multisubunit enzyme with the SecA protein as its peripheral membrane domain and the SecY/E protein as its integral domain. SecB, by binding to proOmpA in the cytosol, prevents its aggregation or association with membranes at nonproductive sites. The SecA receptor binds the proOmpA-SecB complex (Kd approximately 6 x 10(-8) M) through direct recognition of both the SecB (Kd approximately 2 x 10(-7) M) as well as the leader and mature domains of the precursor protein. SecB has a dual function in stabilizing the precursor and in passing it on to membrane-bound SecA, the next step in the pathway. SecA itself is bound to the membrane by its affinity (Kd approximately 4 x 10(-8) M) for SecY/E and for acidic lipids. The functions of SecB and SecA as a two-stage receptor system are linked by their affinity for each other.     

Programming desiccation-tolerance: from plants to seeds to resurrection plants

Desiccation-tolerance (DT) evolved as the key solution to survival on land by the early algal ancestors of terrestrial plants. This 'first' DT involved utilizing rapidly mobilisable repair mechanisms and is still found today in mosses, such as Tortula ruralis, and ferns, such as Mohria caffrorum. The first seed plants lost vegetative DT while investing their seeds with tolerance mechanisms improving their survival in unfavourable environments. The mechanisms of DT in seeds are strongly connected to their developmentally regulated maturation programs. We propose that angiosperm resurrection plants acquired tolerance by re-activating their innate DT mechanisms in their vegetative tissues. Here we review the current hypotheses regarding the genetic evidence for the evolution of DT in resurrection plants. We also present strong evidence showing the activation of seed specific genetic elements in the vegetative tissues of resurrection plants.     

How to prevent losses of protein by adsorption to glass and plastic

An improved procedure for reducing the loss of protein by adsorption to glass or plastic surfaces is reported. For working with proteins at the microgram level, the solvent is modified by adding glycerol (50% final concentration) or Triton X-100 (0.2 mM final concentration). Coating the plastic or glass surfaces with proteins such as bovine serum albumin or other materials is not as effective; adding proteins such as bovine serum albumin to the solvent is counterproductive.     

Resistance to immunization

Despite a well documented efficacy and a generally good adhesion of a large majority of the lay population and health care professionals as well, immunization is still the object of controversies. It affects several vaccines against hepatitis B, influenza, BCG, pertussis and measles. In most cases, polemics on vaccination result from a temporal association between a vaccination and the revelation of a serious disease, when the immunization is performed at the peak of incidence of the disease. Controversies can also be initiated by scientific publications, even though these are often biased, by a misinterpretation or the absence of official positions from the health care authorities, or by justice decisions, whose criteria differ from the scientists' ones and are therefore difficult to understand for the lay public. Furthermore, the scientific demonstration of the exclusion of a risk is difficult to obtain. In the past, it appeared to be very difficult to stop controversies. Improving the communication is the main issue, towards lay public as well as health care professionals. This implies to learn how to better use the media, to motivate and educate professionals who administer vaccine, and anticipate the eventuality of a temporal relationship between vaccination and occurrence of a serious disease by an improved management of the pharmacovigilance system.     

Sensitivity of Saccharomyces cerevisiae to tannic acid is due to iron deprivation

Tannic acid inhibited the growth of the yeast Saccharomyces cerevisiae. Growth medium supplementation with more nitrogen or metal ions showed that only iron ions could restore the maximal growth rate of S. cerevisiae. Tannic acid resistant mutants were previously isolated by screening for tannic acid resistance and were all cytoplasmic petite mutants. While the wild type was very sensitive to iron deprivation conditions when grown in aerobic conditions, the mutants, whether grown aerobically or anaerobically, showed the same growth rate under iron-limited conditions as under iron-repleted conditions. Also, the wild type grown anaerobically was not affected by iron-limited conditions. Cytoplasmic petite mutants obtained by ethidium bromide mutagenesis behaved like the other mutants. During iron limitation, the wild type showed a reduced oxygen uptake rate. Maximal growth rate of the wild type in iron-limited conditions could be restored by the addition to the media of unsaturated fatty acids and sterol. Iron deprivation caused by tannic acid may thus affect the synthesis of a functional respiratory chain as well as the synthesis of unsaturated fatty acids and (or) sterol.     

Reverse tolerance to amphetamine evokes reverse tolerance to 5-hydroxytryptophan

Repeated intermittent administration of amphetamine in mice caused reverse tolerance to 5-hydroxy-L-tryptophan (5-HTP)-induced head twitch, as well as to amphetamine-induced stereotypy. The repeated administration of 5-HTP alone also resulted in reverse tolerance in the head-twitch test. Daily pretreatment with haloperidol prior to amphetamine administration blocked the development of both reverse tolerance to amphetamine and to 5-HTP, whereas daily pretreatment with cyproheptadine prior to amphetamine blocked only the reverse tolerance to 5-HTP. On the other hand, 5-HTP-induced reverse tolerance was blocked by daily pretreatment with cyproheptadine, but not with haloperidol. There appears to be no difference in the persistence of the reverse tolerance to 5-HTP, whether induced by amphetamine or by 5-HTP; in both instances, the persistence does not correlate with the persistence of reverse tolerance to amphetamine. The data suggest that the reverse tolerance to amphetamine and the associated reverse tolerance to 5-HTP are independent events, both of which are mediated by dopaminergic mechanisms.     

Physiological responses to stress with reference to the domestic fowl.

Environmental stress may effect the outcome of routine investigations as well as experimental procedures. This is illustrated by investigations in the domestic fowl.     

Post-irradiation promotes susceptibility to reprogramming to pluripotent state in human fibroblasts

Ionizing radiation causes not only targeted effects in cells that have been directly irradiated but also non-targeted effects in several cell generations after initial exposure. Recent studies suggest that radiation can enrich for a population of stem cells, derived from differentiated cells, through cellular reprogramming. Here, we elucidate the effect of irradiation on reprogramming, subjected to two different responses, using an induced pluripotent stem cell (iPSC) model. iPSCs were generated from non-irradiated cells, directly-irradiated cells, or cells subsequently generated after initial radiation exposure. We found that direct irradiation negatively affected iPSC induction in a dose-dependent manner. However, in the post-irradiated group, after five subsequent generations, cells became increasingly sensitive to the induction of reprogramming compared to that in non-irradiated cells as observed by an increased number of Tra1-81-stained colonies as well as enhanced alkaline phosphatase and Oct4 promoter activity. Comparative analysis, based on reducing the number of defined factors utilized for reprogramming, also revealed enhanced efficiency of iPSC generation in post-irradiated cells. Furthermore, the phenotypic acquisition of characteristics of pluripotent stem cells was observed in all resulting iPSC lines, as shown by morphology, the expression of pluripotent markers, DNA methylation patterns of pluripotency genes, a normal diploid karyotype, and teratoma formation. Overall, these results suggested that reprogramming capability might be differentially modulated by altered radiation-induced responses. Our findings provide that susceptibility to reprogramming in somatic cells might be improved by the delayed effects of non-targeted response, and contribute to a better understanding of the biological effects of radiation exposure.     

Nature of the human immune response to exposure to protein-producing microorganisms

The influence of the live culture and dried biomass of yeast-like fungi of the genus Candida on the characteristics of immune response in 1094 persons exposed to different concentrations of these fungi was studied. The subjects developed sensitization, manifested by immediate and cellular hypersensitivity reactions, as well as by disturbances in T-dependent and humoral immunity. The pattern of the detected changes depended on the concentration of the active factors and on the systemic condition of the subjects. Live fungal cells showed higher antigenic potency and were conducive to the development of allergic symptoms.     

Gangliosides noncovalently bound to DEAE-Sephadex: application to purification of anti-ganglioside antibodies

A simple, rapid, effective, and inexpensive method for the purification of ligands having high affinity for gangliosides has been developed. DEAE-Sephadex has a high capacity for binding gangliosides (approx 1/1.6, w/w). The gangliosides, bound to the support by electrostatic and hydrophobic interactions, showed a high resistance, in an aqueous environment, to being detached by eluants commonly employed to desorb ligands (i.e., low or high pH or chaotropic agent solutions) or by nonionic detergent solutions as well as by organic solvents. The DEAE-Sephadex-ganglioside complex was assayed as an immunoadsorbent for purifying anti-GM1 ganglioside antibodies from serum of an immunized rabbit. The specific activity of the purified antibodies was 200- to 400-fold higher, and the recovery of the anti-ganglioside activity was above 50%, with respect to the untreated antiserum. The preparation of the complex and the purification of the antibodies can be done in less than 5 h. The glycolipids from the complex can be recovered by elution with organic solvents containing salt or volatile base solutions, and reused. In principle, this method can be adapted for other anionic amphipathic receptor molecules to purify ligands which bind to them.