Intrasarcomere [Ca2+] gradients in ventricular myocytes revealed by high speed digital imaging microscopy.

Cardiac muscle contraction is triggered by a small and brief Ca2+ entry across the t-tubular membranes, which is believed to be locally amplified by release of Ca2+ from the adjacent junctional sarcoplasmic reticulum (SR). As Ca2+ diffusion is thought to be markedly attenuated in cells, it has been predicted that significant intrasarcomeric [Ca2+] gradients should exist during activation. To directly test for this, we measured [Ca2+] distribution in single cardiac myocytes using fluorescent [Ca2+] indicators and high speed, three-dimensional digital imaging microscopy and image deconvolution techniques. Steep cytosolic [Ca2+] gradients from the t-tubule region to the center of the sarcomere developed during the first 15 ms of systole. The steepness of these [Ca2+] gradients varied with treatments that altered Ca2+ release from internal stores. Electron probe microanalysis revealed a loss of Ca2+ from the junctional SR and an accumulation, principally in the A-band during activation. We propose that the prolonged existence of [Ca2+] gradients within the sarcomere reflects the relatively long period of Ca2+ release from the SR, the localization of Ca2+ binding sites and Ca2+ sinks remote from sites of release, and diffusion limitations within the sarcomere. The large [Ca2+] transient near the t-tubular/ junctional SR membranes is postulated to explain numerous features of excitation-contraction coupling in cardiac muscle.

Kinetic equilibrium of forces and molecular events in muscle contraction

In biomolecular systems, the mechanical transfer of free energy occurs with both high efficiency and high speed. It is shown here that such a transfer can be achieved only if the participating free-energy-storing elements exhibit opposing relationships between their content of free energy and the force they exert in the transfer direction. A kinetic equilibrium of forces (KEF) results, in which the transfer of free energy is mediated essentially by thermal molecular motion. On the basis of present evidence, KEF is used as a guiding principle in developing a mechanical model of the crossbridge cycle in muscle contraction. The model allows the basic features of molecular events to be visualized in terms of plausible structures. Real understanding of the process will require identification of the elements that perform the functions described here. Besides chemomechanical energy transduction, KEF may have a role in other biomolecular processes in which free energy is transferred mechanically over large distances.

Field theory in superfluid 3He: What are the lessons for particle physics, gravity, and high-temperature superconductivity?

There are several classes of homogeneous Fermi systems that are characterized by the topology of the energy spectrum of fermionic quasiparticles: (i) gapless systems with a Fermi surface, (ii) systems with a gap in their spectrum, (iii) gapless systems with topologically stable point nodes (Fermi points), and (iv) gapless systems with topologically unstable lines of nodes (Fermi lines). Superfluid 3He-A and electroweak vacuum belong to the universality class 3. The fermionic quasiparticles (particles) in this class are chiral: they are left-handed or right-handed. The collective bosonic modes of systems of class 3 are the effective gauge and gravitational fields. The great advantage of superfluid 3He-A is that we can perform experiments by using this condensed matter and thereby simulate many phenomena in high energy physics, including axial anomaly, baryoproduction, and magnetogenesis. 3He-A textures induce a nontrivial effective metrics of the space, where the free quasiparticles move along geodesics. With 3He-A one can simulate event horizons, Hawking radiation, rotating vacuum, etc. High-temperature superconductors are believed to belong to class 4. They have gapless fermionic quasiparticles with a “relativistic” spectrum close to gap nodes, which allows application of ideas developed for superfluid 3He-A.

Low- and high-level transgenic expression of β2-adrenergic receptors differentially affect cardiac hypertrophy and function in Gαq-overexpressing mice

Transgenic overexpression of Gαq in the heart triggers events leading to a phenotype of eccentric hypertrophy, depressed ventricular function, marked expression of hypertrophy-associated genes, and depressed β-adrenergic receptor (βAR) function. The role of βAR dysfunction in the development of this failure phenotype was delineated by transgenic coexpression of the carboxyl terminus of the βAR kinase (βARK), which acts to inhibit the kinase, or concomitant overexpression of the β2AR at low (≈30-fold, Gαq/β2ARL), moderate (≈140-fold, Gαq/β2ARM), and high (≈1,000-fold, Gαq/β2ARH) levels above background βAR density. Expression of the βARK inhibitor had no effect on the phenotype, consistent with the lack of increased βARK levels in Gαq mice. In marked contrast, Gαq/β2ARL mice displayed rescue of hypertrophy and resting ventricular function and decreased cardiac expression of atrial natriuretic factor and α-skeletal actin mRNA. These effects occurred in the absence of any improvement in basal or agonist-stimulated adenylyl cyclase (AC) activities in crude cardiac membranes, although restoration of a compartmentalized β2AR/AC signal cannot be excluded. Higher expression of receptors in Gαq/β2ARM mice resulted in salvage of AC activity, but hypertrophy, ventricular function, and expression of fetal genes were unaffected or worsened. With ≈1,000-fold overexpression, the majority of Gαq/β2ARH mice died with cardiomegaly at 5 weeks. Thus, although it appears that excessive, uncontrolled, or generalized augmentation of βAR signaling is deleterious in heart failure, selective enhancement by overexpressing the β2AR subtype to limited levels restores not only ventricular function but also reverses cardiac hypertrophy.

World food and agriculture: Outlook for the medium and longer term

The world has been making progress in improving food security, as measured by the per person availability of food for direct human consumption. However, progress has been very uneven, and many developing countries have failed to participate in such progress. In some countries, the food security situation is today worse than 20 years ago. The persistence of food insecurity does not reflect so much a lack of capacity of the world as a whole to increase food production to whatever level would be required for everyone to have consumption levels assuring satisfactory nutrition. The world already produces sufficient food. The undernourished and the food-insecure persons are in these conditions because they are poor in terms of income with which to purchase food or in terms of access to agricultural resources, education, technology, infrastructure, credit, etc., to produce their own food. Economic development failures account for the persistence of poverty and food insecurity. In the majority of countries with severe food-security problems, the greatest part of the poor and food-insecure population depend greatly on local agriculture for a living. In such cases, development failures are often tantamount to failures of agricultural development. Development of agriculture is seen as the first crucial step toward broader development, reduction of poverty and food insecurity, and eventually freedom from excessive economic dependence on poor agricultural resources. Projections indicate that progress would continue, but at a pace and pattern that would be insufficient for the incidence of undernutrition to be reduced significantly in the medium-term future. As in the past, world agricultural production is likely to keep up with, and perhaps tend to exceed, the growth of the effective demand for food. The problem will continue to be one of persistence of poverty, leading to growth of the effective demand for food on the part of the poor that would fall short of that required for them to attain levels of consumption compatible with freedom from undernutrition.

Two Structurally Similar Maize Cytosolic Superoxide Dismutase Genes, Sod4 and Sod4A, Respond Differentially to Abscisic Acid and High Osmoticum1

The maize (Zea mays) superoxide dismutase genes Sod4 and Sod4A are highly similar in structure but each responds differentially to environmental signals. We examined the effects of the hormone abscisic acid (ABA) on the developmental response of Sod4 and Sod4A. Although both Sod4 and Sod4A transcripts accumulate during late embryogenesis, only Sod4 is up-regulated by ABA and osmotic stress. Accumulation of Sod4 transcript in response to osmotic stress is a consequence of increased endogenous ABA levels in developing embryos. Sod4 mRNA is up-regulated by ABA in viviparous-1 mutant embryos. Sod4 transcript increases within 4 h with ABA not only in developing embryos but also in mature embryos and in young leaves. Sod4A transcript is up-regulated by ABA only in young leaves, but neither Sod4 nor Sod4A transcripts changed in response to osmotic stress. Our data suggest that in leaves Sod4 and Sod4A may respond to ABA and osmotic stress via alternate pathways. Since the Sod genes have a known function, we hypothesize that the increase in Sod mRNA in response to ABA is due in part to ABA-mediated metabolic changes leading to changes in oxygen free radical levels, which in turn lead to the induction of the antioxidant defense system.

Lambert-Eaton sera reduce low-voltage and high-voltage activated Ca2+ currents in murine dorsal root ganglion neurons.

Voltage-gated Ca2+ channels are categorized as either high-voltage activated (HVA) or low-voltage activated (LVA), and a subtype (or subtypes) of HVA Ca2+ channels link the presynaptic depolarization to rapid neuro-transmitter release. Reductions in transmitter release are characteristic of the autoimmune disorder, Lambert-Eaton syndrome (LES). Because antibodies from LES patients reduce Ca2+ influx in a variety of cell types and disrupt the intramembrane organization of active zones at neuromuscular synapses, specificity of LES antibodies for the Ca2+ channels that control transmitter release has been suggested as the mechanism for disease. We tested sera from four patients with LES. Serum samples from three of the four patients reduced both the maximal LVA and HVA Ca2+ conductances in murine dorsal root ganglion neurons. Thus, even though LES is expressed as a neuromuscular and autonomic disorder, our studies suggest that Ca2+ channels may be broadly affected in LES patients. To account for the specificity of disease expression, we suggest that incapacitation of only a fraction of the Ca2+ channels clustered at active zones would severely depress transmitter release. In particular, if several Ca2+ channels in a cluster are normally required to open simultaneously before transmitter release becomes likely, the loss of a few active zone Ca2+ channels would exponentially reduce the probability of transmitter release. This model may explain why LES is expressed as a neuromuscular disorder and can account for a clinical hallmark of LES, facilitation of neuromuscular transmission produced by vigorous voluntary effort.

Specific and high-affinity binding of inositol phosphates to an isolated pleckstrin homology domain.

Pleckstrin homology (PH) domains are found in many signaling molecules and are thought to be involved in specific intermolecular interactions. Their binding to several proteins and to membranes containing 1-alpha-phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] has been reported. A region that includes the PH domain has also been implicated in binding of phospholipase C-delta 1 (PLC-delta 1) to both PtdIns(4,5)P2 and D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] [Cifuentes, M. E., Delaney, T. & Rebecchi, M. J. (1994) J. Biol. Chem. 269, 1945-1948]. We report herein that the isolated PH domain from PLC-delta 1 binds to both PtdIns(4,5)P2 and Ins(1,4,5)P3 with high affinity and shows the same binding specificity seen by others with whole PLC-delta 1. Thus the PH domain is functionally and structurally modular. These results demonstrate stereo-specific high-affinity binding by an isolated PH domain and further support a functional role for PH domains in the regulation of PLC isoforms. Other PH domains did not bind strongly to the compounds tested, suggesting that inositol phosphates and phospholipids are not likely physiological ligands for all PH domains. Nonetheless, since all PH-domain-containing proteins are associated with membrane surfaces, several PH domains bind to specific sites on membranes, and PH domains appear to be electrostatically polarized, a possible general role for PH domains in membrane association is suggested.

Structure and function of a human transcription factor TFIIIB subunit that is evolutionarily conserved and contains both TFIIB- and high-mobility-group protein 2-related domains.

Transcription factor TFIIIB plays a central role in transcription initiation by RNA polymerase III on genes encoding tRNA, 5S rRNA, and other small structural RNAs. We report the purification of a human TFIIIB-derived complex containing only the TATA-binding polypeptide (TBP) and a 90-kDa subunit (TFIIIB90) and the isolation of a cDNA clone encoding the 90-kDa subunit. The N-terminal half of TFIIIB90 exhibits sequence similarity to the yeast TFIIIB70 (BRF) and the class II transcription factor TFIIB and interacts weakly with TBP. The C-terminal half of TFIIIB90 contains a high-mobility-group protein 2 (HMG2)-related domain and interacts strongly with TBP. Recombinant TFIIIB90 plus recombinant human TBP substitute for human TFIIIB in a complementation assay for transcription of 5S, tRNA, and VA1 RNA genes, and both the TFIIB-related domain and the HMG2-related domain are required for this activity. TFIIIB90 is also required for transcription of human 7SK and U6 RNA genes by RNA polymerase III, but apparently within a complex distinct from the TBP/TFIIIB90 complex.