Expression and biochemical analysis of the entire HIV-2 gp41 ectodomain: determinants of stability map to N- and C-terminal sequences outside the 6-helix bundle core

The folding of HIV gp41 into a 6-helix bundle drives virus-cell membrane fusion. To examine the structural relationship between the 6-helix bundle core domain and other regions of gp41, we expressed in Escherichia coli, the entire ectodomain of HIV-2(ST) gp41 as a soluble, trimeric maltose-binding protein (MBP)/gp41 chimera. Limiting proteolysis indicated that the Cys-591-Cys-597 disulfide-bonded region is outside a core domain comprising two peptides, Thr-529-Trp-589 and Val-604-Ser-666. A biochemical examination of MBP/gp41 chimeras encompassing these core peptides; indicated that the N-terminal polar segment, 521-528, and C-terminal membrane-proximal segment, 658-666, cooperate in stabilizing the ectodomain. A functional interaction between sequences outside the gp41 core may contribute energy to membrane fusion. (C) 2004 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.

theta-defensins prevent HIV-1 Env-mediated fusion by binding gp41 and blocking 6-helix bundle formation

Retrocyclin-1, a 0-defensin, protects target cells from human immunodeficiency virus, type 1 (HIV-1) by preventing viral entry. To delineate its mechanism, we conducted fusion assays between susceptible target cells and effector cells that expressed HIV-1 Env. Retrocyclin-1 (4 mu M) completely blocked fusion mediated by HIV-1 Envs that used CXCR4 or CCR5 but had little effect on cell fusion mediated by HIV-2 and simian immunodeficiency virus Envs. Retrocyclin-1 inhibited HIV-1 Env-mediated fusion without impairing the lateral mobility of CD4, and it inhibited the fusion of CD4-deficient cells with cells bearing CD4-independent HIV-1 Env. Thus, it could act without cross-linking membrane proteins or inhibiting gp120-CD4 interactions. Retrocyclin-1 acted late in the HIV-1 Env fusion cascade but prior to 6-helix bundle formation. Surface plasmon resonance experiments revealed that retrocyclin bound the ectodomain of gp41 with high affinity in a glycan-independent manner and that it bound selectively to the gp41 C-terminal heptad repeat. Native-PAGE, enzyme-linked immunosorbent assay, and CD spectroscopic analyses all revealed that retrocyclin-1 prevented 6-helix bundle formation. This mode of action, although novel for an innate effector molecule, resembles the mechanism of peptidic entry inhibitors based on portions of the gp41 sequence.

Correlation between carbon isotope discrimination and transpiration efficiency in lines of the C-4 species Sorghum bicolor in the glasshouse and the field

Transpiration efficiency, W, the ratio of plant carbon produced to water transpired and carbon isotope discrimination of leaf dry matter, Delta(d)' were measured together on 30 lines of the C-4 species, Sorghum bicolor in the glasshouse and on eight lines grown in the field. In the glasshouse, the mean W observed was 4.9 mmol C mol(-1) H2O and the range was 0.8 mmol C mol(-1) H2O The mean Delta(d) was 3.0 parts per thousand and the observed range was 0.4 parts per thousand. In the field, the mean W was lower at 2.8 mmol C mol H2O and the mean Delta(d) was 4.6 parts per thousand. Significant positive correlations between W and Delta(d) were observed for plants grown in the glasshouse and in the field. The observed correlations were consistent with theory, opposite to those for C-4 species, and showed that variation in Delta(d) was an integrated measure of long-term variation in the ratio of intercellular to ambient CO2 partial pressure, p(i)/p(a). Detailed gas exchange measurements of carbon isotope discrimination during CO2 uptake, Delta(A) and p(i)/p(a) were made on leaves of eight S. bicolor lines. The observed relationship between Delta(A) and p(i)/p(a) was linear with a negative slope of 3.7 parts per thousand in Delta(A) for a unit change in p(i)/p(a). The slope of this linear relationship between Delta(A) and p(i)/p(a) in C-4 species is dependent on the leakiness of the CO2 concentrating mechanism of the C pathway, We estimated the leakiness (defined as the fraction of CO2 released in the bundle sheath by C-4 acid decarboxylations, which is lost by leakage) to be 0.2. We conclude that, although variation in Delta(d) observed in the 30 lines of S. bicolor is smaller than that commonly observed in C-4 species, it also reflects variation in transpiration efficiency, W. Among the eight lines examined in detail and in the environments used, there was considerable genotype x environment interaction.

Small molecular probes for G-protein-coupled C5a receptors: Conformationally constrained antagonists derived from the C terminus of the human plasma protein C5a

Activation of the human complement system of plasma proteins in response to infection or injury produces a 4-helix bundle glycoprotein (74 amino acids) known as C5a. C5a binds to G-protein-coupled receptors on cell surfaces triggering receptor-ligand internalization, signal transduction, and powerful inflammatory responses. Since excessive levels of C5a are associated with autoimmune and chronic inflammatory disorders, inhibitors of receptor activation may have therapeutic potential. We now report solution structures and receptor-binding and antagonist activities for some of the first small molecule antagonists of C5a derived from its hexapeptide C terminus. The antagonist NMe-Phe-Lys-Pro-D-Cha-Trp-D-Arg-CO2H (1) surprisingly shows an unusually well-defined solution structure as determined by H-1 NMR spectroscopy. This is one of the smallest acyclic peptides found to possess a defined solution conformation, which can be explained by the constraining role of intramolecular hydrogen bonding. NOE and coupling constant data, slow deuterium exchange, and a low dependence on temperature for the chemical shift of the D-Cha-NH strongly indicate an inverse gamma turn stabilized by a D-Cha-NH ... OC-Lys hydrogen bond. Smaller conformational populations are associated with a hydrogen bond between Trp-NH ... OC-Lys, defining a type II beta turn distorted by the inverse gamma turn incorporated within it. An excellent correlation between receptor-affinity and antagonist activity is indicated for a limited set of synthetic peptides. Conversion of the C-terminal carboxylate of 1 to an amide decreases antagonist potency 5-fold, but potency is increased up to 10-fold over 1 if the amide bond is made between the C-terminal carboxylate and a Lys/Orn side chain to form a cyclic analogue. The solution structure of cycle 6 also shows gamma and beta turns; however, the latter occurs in a different position, and there are clear conformational changes in 6 vs 1 that result in enhanced activity. These results indicate that potent C5a antagonists can be developed by targeting site 2 alone of the C5a receptor and define a novel pharmacophore for developing powerful receptor probes or drug candidates.

Refining the risk-benefit equation for thrombolysis: How to identity the low risk patient before administering thrombolytic therapy

In view of the relative risk of intracranial haemorrhage and major bleeding with thrombolytic therapy, it is important ro identify as early as possible the low risk patient who may not have a net clinical benefit from thrombolysis in the setting of acute myocardial infarction. An analysis of 5434 hospital-treated patients with myocardial infarction in the Perth MONICA study showed that age below 60 and absence of previous infarction or diabetes, shock, pulmonary oedema, cardiac arrest and Q-wave or left bundle branch block on the initial ECG identified a large group of patients with a 28 day mortality of only 1%, and one year mortality of only 2%. Identification of baseline risk in this way helps refine the risk-benefit equation for thrombolytic therapy, and may help avoid unnecessary use of thrombolysis in those unlikely to benefit.

Asymptotic limits of a self-dual Ginzburg-Landau functional in bounded domains

In the author's joint paper [HJS] with Jest and Struwe, we discuss asymtotic limits of a self-dual Ginzburg-Landau functional involving a section of a line bundle over a closed Riemann surface and a connection on this bundle. In this paper, the author generalizes the above results [HJS] to the case of bounded domains.

Thylakoid membrane architecture

Confocal scanning laser microscopic observations were made on live chloroplasts in intact cells and on mechanically isolated, intact chloroplasts. Chlorophyll fluorescence was imaged to observe thylakoid membrane architecture. C-3 plant species studied included Spinacia oleracea L., Spathiphyllum sp. Schott, cv. 'Mauna Loa', and Pisum sativum L. C-4 plants were also investigated: Saccharum officinarum L., Sorghum bicolor L. Moench, Zea mays L. and Panicum miliaceum L. Some Spinacia chloroplasts were treated with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) to enhance or sodium dithionite (SD) to reduce the photosystem II fluorescence signal. Confocal microscopy images of C-3 chloroplasts differed from electron microscopy pictures because they showed discrete spots of bright fluorescence with black regions between them. There was no evidence of fluorescence from stroma thylakoids. The thylakoid membrane system at times appeared to be string-like, with brightly fluorescing grana lined up like beads. C-4 bundle sheath chloroplasts were imaged from three different types of C-4 plants. Saccharum and Sorghum bundle sheath chloroplasts showed homogeneous fluorescence and were much dimmer than mesophyll chloroplasts. Zea had rudimentary grana, and dim, homogeneous intergrana fluorescence was visualised. Panicum contained thylakoids similar in appearance and string-like arrangement to mesophyll chloroplasts. Isolated Pisum chloroplasts, treated with a drop of 5 mM MgCl2 showed a thylakoid membrane system which appeared to be unravelling. Spongy mesophyll chloroplasts of Spinacia treated with 5 mM sodium dithionite showed a granal thylakoid system with distinct regions of no fluorescence. A time-series experiment provided evidence of dynamic membrane rearrangements over a period of half an hour.

Heat flow for Yang-Mills-Higgs fields, part I

The Yang-Mills-Higgs field generalizes the Yang-Mills field. The authors establish the local existence and uniqueness of the weak solution to the heat flow for the Yang-Mills-Higgs field in a vector bundle over a compact Riemannian 4-manifold, and show that the weak solution is gauge-equivalent to a smooth solution and there are at most finite singularities at the maximum existing time.

Heat flow for the Yang-Mills-Higgs field and the Hermitian Yang-Mills-Higgs metric

For a parameter lambda > 0, we study a type of vortex equations, which generalize the well-known Hermitian-Einstein equation, for a connection A and a section phi of a holomorphic vector bundle E over a Kahler manifold X. We establish a global existence of smooth solutions to heat flow for a self-dual Yang-Mills-Higgs field on E. Assuming the lambda -stability of (E, phi), we prove the existence of the Hermitian Yang-Mills-Higgs metric on the holomorphic bundle E by studying the limiting behaviour of the gauge flow.

The effects of moderate and low levels of acoustic overstimulation on stereocilia and their tip links in the guinea pig

Guinea pigs were exposed to pure tones of 10 kHz at intensities between 98 and 115 dB SPL for 5-30 min, to produce varying degrees of acoustic trauma. Changes in auditory thresholds were measured electrophysiologically, and the animals were immediately fixed for scanning electron microscopy. Correlation between morphological changes to the hair bundle and losses in threshold, showed that with the smallest degrees of trauma (98 dB SPL for 15 min, mean maximum threshold loss of 22 dB), damage was confined to a small stretch of inner hair cells (IHC), with only subtle changes to the stereocilia of the outer hair cells (OHC). At exposure intensities greater than 102 dB SPL (duration: 15 min) the IHC stereocilia in the centre of the lesion were always substantially disarrayed. Substantial damage to the OHC bundles was seen only with exposures above 110 dB SPL(duration: greater than or equal to 5 min), producing threshold losses of 50 dB or more. Tip links were lost only where the stereocilia were disarrayed. It is concluded that the tip links are not the most vulnerable components of the cochlear hair cell, but that relatively low levels of acoustic stimulation can cause significant damage to the stereociliary bundle of the IHCs.

Comparative sperm ultrastructure in five genera of the nudibranch family Chromodorididae (Gastropoda : Opisthobranchia)

Sperm ultrastructure is examined in representatives of five genera of the nudibranch gastropod family Chromodorididae: (Chromodoris, Hypselodoris, Glossodoris, Risbecia and Pectenodoris) and the results compared with previous work on other gastropods, especially other nudibranchs. As chromodoridid phylogeny is still incompletely understood, this study partly focuses on the search for new and as yet untapped sources of informative characters. Like spermatozoa of most other heterobranch gastropods, those of the Chromodorididae are elongate, complex cells composed of an acrosomal complex (small, rounded acrosomal vesicle, and columnar acrosomal pedestal), a condensed nucleus, sub-nuclear ring, a highly modified mid-piece (axoneme + coarse fibres surrounded by a glycogen-containing, helically-coiled mitochondrial derivative) and terminally a glycogen piece (or homologue thereof). The finely striated acrosomal pedestal is a synapomorphy of all genera examined here, but interestingly also occurs in at least one dorid (Rostanga arbutus). Substantial and potentially taxonomically informative differences were also observed between genera in the morphology of the nucleus, the neck region of the mid-piece, and also the terminal glycogen piece. The subnuclear ring is shown for the first time to be a segmented, rather than a continuous structure; similarly, the annular complex is shown to consist of two structures, the annulus proper and the herein-termed annular accessory body.