Lucigenin luminescence as a measure of intracellular superoxide dismutase activity in Escherichia coli

Lucigenin and paraquat are similar in that each can be taken into Escherichia coli and can then mediate O₂ production by cycles of univalent reduction, to the corresponding monocation radical, followed by autoxidation. Thus, both compounds caused induction of enzymes that are regulated by the soxRS regulon. The lucigenin cation radical has the added property of reacting with O₂, in a radical–radical addition, to yield an unstable dioxetane, whose decomposition yields light. Superoxide dismutases (SOD), by decreasing [O₂], inhibit light production and to the same degree inhibit other O₂-dependent reactions in the cell. Lucigenin luminescence was used to show that the levels of SOD in the parental strain provide ≈95% protection of all O₂-sensitive targets in E. coli. This degree of protection was so close to the limit of 100% that halving the parental level of [SOD], or increasing it 5-fold, had only marginal effects on the intensity of lucigenin-dependent luminescence.     

Specific atomic groups and RNA helix geometry in acceptor stem recognition by a tRNA synthetase

Oligonucleotides that recapitulate the acceptor stems of tRNAs are substrates for aminoacylation by many tRNA synthetases in vitro, even though these substrates are missing the anticodon trinucleotides of the genetic code. In the case of tRNA^Ala a single acceptor stem GU base pair at position 3·70 is essential, based on experiments where the wobble pair has been replaced by alternatives such as IU, GC, and AU, among others. These experiments led to the conclusion that the minor-groove free 2-amino group (of guanosine) of the GU wobble pair is essential for charging. Moreover, alanine-inserting tRNAs (amber suppressors) that replace GU with mismatches such as GA and CA are partially active in vivo and can support growth of an Escherichia coli tRNA^Ala knockout strain, leading to the hypothesis that a helix irregularity and nucleotide functionalities are important for recognition. Herein we investigate the charging in vitro of oligonucleotide and full-length tRNA substrates that contain mismatches at the position of the GU pair. Although most of these substrates have undetectable activity, GA and CA variants retain some activity, which is, nevertheless, reduced by at least 100-fold. Thus, the in vivo assays are much less sensitive to large changes in aminoacylation kinetic efficiency of 3·70 variants than is the in vitro assay system. Although these functional data do not clarify all of the details, it is now clear that specific atomic groups are substantially more important in determining kinetic efficiency than is a helical distortion. By implication, the activity of mutant tRNAs measured in the in vivo assays appears to be more dependent on factors other than aminoacylation kinetic efficiency.     

Primary CD8+ cells from HIV-infected individuals can suppress productive infection of macrophages independent of β-chemokines

The productive infection of human monocyte-derived macrophages (M) by HIV was suppressed by primary CD8⁺ cells from asymptomatic HIV-infected individuals. This anti-HIV response was noncytotoxic; removal of the CD8⁺ cells from the infected M leads to virus production. CD8⁺ cells inhibited HIV replication when separated from the infected M by a transwell filter insert, indicating a diffusible factor made by the CD8⁺ cells suppressed productive infection of M. Three β-chemokines, which can be secreted by activated CD8⁺ cells, RANTES (regulated on activation normal T cell expressed and secreted), macrophage inflammatory protein (MIP)-1α and MIP-1β prevented HIV replication in the M cultures. In addition, incubation of acutely infected M with a mixture of neutralizing antibodies to RANTES, MIP-1α, and MIP-1β enhanced virus replication. Nevertheless, neutralization of β-chemokines with specific antibodies did not abolish the suppression by CD8⁺ cells of HIV replication in M. Thus, even though β-chemokines decrease HIV replication in M, these cytokines are not responsible for the ability of CD8⁺ cells to inhibit HIV production in these cells.     

Specific requirement for CD3ɛ in T cell development

T cell antigen receptor (TCR) and pre-TCR complexes are composed of clonotypic heterodimers in association with dimers of signal transducing invariant subunits (CD3γ, -δ, -, and ζ). The role of individual invariant subunits in T cell development has been investigated by generating gene-specific mutations in mice. Mutation of CD3γ, -δ, or ζ results in an incomplete block in development, characterized by reduced numbers of mature T cells that express low levels of TCR. In contrast, mature T cells are absent from CD3^−/− mice, and thymocyte development is arrested at the early CD4⁻CD8⁻ stage. Although these results suggest that CD3 is essential for pre-TCR and TCR expression/function, their interpretation is complicated by the fact that expression of the CD3γ and CD3δ genes also is reduced in CD3^−/− mice. Thus, it is unclear whether the phenotype of CD3^−/− mice reflects the collective effects of CD3γ, -δ, and - deficiency. By removing the selectable marker (PGK-NEO) from the targeted CD3 gene via Cre/loxP-mediated recombination, we generated mice that lack CD3 yet retain normal expression of the closely linked CD3γ and CD3δ genes. These (CD3^Δ/Δ) mice exhibited an early arrest in T cell development, similar to that of CD3^−/− mice. Moreover, the developmental defect could be rescued by expression of a CD3 transgene. These results identify an essential role for CD3 in T cell development not shared by the CD3γ, CD3δ, or ζ-family proteins and provide further evidence that PGK-NEO can influence the expression of genes in its proximity.     

Aminoguanidine prevents age-related arterial stiffening and cardiac hypertrophy

Aging is associated with cardiac hypertrophy and arterial stiffening possibly associated with accumulation of advanced glycation end products (AGEs). We evaluated the effect of aminoguanidine, an inhibitor of AGE production, on end-stage alterations of renal and cardiovascular systems. Normotensive WAG/Rij rats were treated from 24 to 30 mo with aminoguanidine and compared with a control group. Aminoguanidine did not modify body and kidney weights but prevented the age-related cardiac hypertrophy (heart weight: 1276 ± 28 mg and 1896 ± 87 mg in 24- and 30-mo-old control animals and 1267 ± 60 mg in 30-mo-old treated rats, P < 0.01). The increase in mesangial surface in aging rats was reduced by 30% by aminoguanidine. Collagen content of the arterial wall increased between 24 and 30 mo whereas elastin content, media thickness, and smooth muscle cell number remained unchanged. Aminoguanidine did not affect these parameters; however, the age-related increase in aortic impedance (12.4 ± 1.4 and 18.2 ± 1.9 10³dyneseccm⁻⁵ in control 24- and 30-mo-old rats, P < 0.01) and the decrease in carotid distensibility (0.79 ± 0.11 and 0.34 ± 0. 07 mm Hg⁻¹ in control 24- and 30-mo-old rats, P < 0.01) were prevented by aminoguanidine. The prevention of arterial stiffening and cardiac hypertrophy in the absence of changes in collagen and elastin content suggests that the effect of aminoguanidine is related to a decrease in the AGE-induced cross-linking of the extracellular matrix.     

Mast cell tumor necrosis factor α production is regulated by MEK kinases

Mast cells synthesize and secrete specific cytokines and chemokines which play an important role in allergic inflammation. Aggregation of the high-affinity Fc receptor (FcRI) for immunoglobulin E (IgE) in MC/9 mouse mast cells stimulates the synthesis and secretion of tumor necrosis factor α (TNF-α). FcRI aggregation activates several sequential protein kinase pathways, leading to increased activity of extracellular signal-regulated kinases (ERKs), c-Jun amino-terminal kinases (JNKs), and the p38 mitogen-activated protein (MAP) kinase. Inhibition of ERKs with the compound PD 098059 had little effect on FcRI-stimulated TNF-α production. Aggregation of FcRI stimulated MEK kinase 1 (MEKK1) activity, which activates JNK kinase (JNKK), the kinase that phosphorylates and activates JNKs. Expression of activated MEKK1 (ΔMEKK1) in MC/9 cells strongly stimulated JNK activity but only weakly stimulated p38 activity, and it induced a large activation of TNF-α promoter-regulated luciferase gene expression. Inhibitory mutant JNK2 expressed in MC/9 cells significantly blunted FcRI stimulation of TNF-α promoter-driven luciferase expression. Wortmannin, an inhibitor of phosphatidylinositol 3-kinase, diminished FcRI-mediated TNF-α synthesis, significantly blunted JNK activation and TNF-α promoter-driven luciferase expression, and only weakly inhibited p38 kinase activation. Inhibition of NFκB activation resulting from ΔMEKK1 expression or FcRI stimulation did not affect TNF-α promoter-driven luciferase expression. Our findings define a MEKK-regulated JNK pathway activated by FcRI that regulates TNF-α production in mast cells.     

Measurement and modeling of the transient difference between blood and subcutaneous glucose concentrations in the rat after injection of insulin

The kinetics of the fall in subcutaneous fluid glucose concentration in anesthetized rats (n = 7) after intravenous injection of insulin (0.5 units/kg) was studied by using 5 × 10⁻⁴ cm² active area, <150-sec 10–90% response time, amperometric glucose sensors. The onset of the decline in the subcutaneous glucose concentration was delayed and statistically different (P < 0.001) from that in blood (8.9 ± 2.1 min vs. 3.3 ± 0.5 min). Similarly, the rate of drop in glucose concentration between 6 and 20 min after the insulin injection was different for subcutaneous tissue (3.9 ± 1.3 mgdl⁻¹ min⁻¹) and blood (6.8 ± 2.0 mgdl⁻¹min⁻¹) (P = 0.003). The hypoglycemic nadir in subcutaneous fluid occurred 24.5 ± 6.8 min after that in the blood (P < 0.001). A “forward” mass-transfer model, predicting the subcutaneous glucose concentration from the blood glucose concentrations and an “inverse” model, predicting the blood glucose concentration from the subcutaneous glucose concentration were derived. By using an algorithm based on the latter, the average discrepancy between the measured blood glucose concentration and that estimated from the subcutaneous measurement through the entire 4-hr experiment was reduced from 22.9% to 11.1% (P = 0.025). The maximum discrepancy during the 40-min period after the injection of insulin was reduced from 84.1% to 29.3% (P = 0.006).     

Enhanced proteolysis of thiopurine S-methyltransferase (TPMT) encoded by mutant alleles in humans (TPMT∗3A, TPMT∗2): Mechanisms for the genetic polymorphism of TPMT activity

TPMT is a cytosolic enzyme that catalyzes the S-methylation of aromatic and heterocyclic sulfhydryl compounds, including medications such as mercaptopurine and thioguanine. TPMT activity exhibits autosomal codominant genetic polymorphism, and patients inheriting TPMT deficiency are at high risk of potentially fatal hematopoietic toxicity. The most prevalent mutant alleles associated with TPMT deficiency in humans have been cloned and characterized (TPMT2 and TPMT3A), but the mechanisms for loss of catalytic activity have not been elucidated. In the present study, we established that erythrocyte TPMT activity was significantly related to the amount of TPMT protein on Western blots of erythrocytes from patients with TPMT activities of 0.4-23 units/ml pRBC (r_s = 0.99; P < 0.001). Similarly, heterologous expression of wild-type (TPMT1) and mutant (TPMT2 and TPMT3A) human cDNAs in yeast and COS-1 cells demonstrated comparable levels of TPMT mRNA but significantly lower TPMT protein with the mutant cDNAs. Rates of protein synthesis were comparable for wild-type and mutant proteins expressed in yeast and with in vitro translation in rabbit reticulocyte lysates. In contrast, pulse–chase experiments revealed significantly shorter degradation half-lives for TPMT2 and TPMT3A (~0.25 hr) compared with wild-type TPMT1 (18 hr). The degradation of mutant proteins was impaired by ATP depletion and in yeast with mutant proteasomes (pre-1 strain) but unaffected by the lysosomal inhibitor chloroquine. These studies establish enhanced degradation of TPMT proteins encoded by TPMT2 and TPMT3A as mechanisms for lower TPMT protein and catalytic activity inherited by the predominant mutant alleles at the human TPMT locus.     

Direct observation of reaction intermediates for a well defined heterogeneous alkene metathesis catalyst

Grafting of [W(NAr)(=CHtBu)(2,5-Me₂NC₄H₂)₂] on a silica partially dehydroxylated at 700°C (SiO_{2- (700)}) generates the corresponding monosiloxy complex [(SiO)W(NAr)(=CHtBu)(2,5-Me₂NC₄H₂)] as the major species (≈90%) along with [(SiO)W(NAr)(CH₂tBu)(2,5-Me₂NC₄H₂)₂], according to mass balance analysis, IR, and NMR studies. This heterogeneous catalyst displays good activity and stability in the metathesis of propene. Very importantly, solid state NMR spectroscopy allows observation of the propagating alkylidene as well as stable metallacyclobutane intermediates. These species have the same reactivity as the initial surface complex [(SiO)W(NAr)(=CHtBu)(2,5-Me₂NC₄H₂)], which shows that they are the key intermediates of alkene metathesis.     

Spectral theory for domains in R of finite measure

Let Ω be a measurable subset of Rⁿ of finite positive Lebesgue measures. The following two problems are considered: (i) Find commuting self-adjoint extensions of the minimal operators -i/x_k, k = 1,..., n (Ω open). (ii) Find a set Λ Rⁿ such that the functions e_λ = exp(iλ₁x₁ +... + iλ_nx_n) for λ Λ form an orthonormal basis for L₂(Ω). The problems are known to be equivalent under mild regularity conditions on Ω, and existence holds in two cases: (i) there is a connected open set Ω′ such that the symmetric difference ΩΔΩ′ is a null set and Ω′ is a fundamental domain for a discrete total subgroup; and (ii)Ω = [unk]_aR (a + [unk]), disjoint union neglecting null sets, in which [unk] is a fundamental domain and R is a “set of representors” for a finite group of translations. Case i is equivalent to a function theoretic condition of Forelli, and case ii is established when the existence of a discrete covariance group is assumed. Generalizations of the geometric results i and ii for spectral sets in arbitrary Lie groups are indicated.     

INAUGURAL ARTICLE by a Recently Elected Academy Member:Charge transfer and transport in DNA

We explore charge migration in DNA, advancing two distinct mechanisms of charge separation in a donor (d)–bridge ({B_j})–acceptor (a) system, where {B_j} = B₁,B₂,… , B_N are the N-specific adjacent bases of B-DNA: (i) two-center unistep superexchange induced charge transfer, d*{B_j}a → d{B_j}a^±, and (ii) multistep charge transport involves charge injection from d* (or d⁺) to {B_j}, charge hopping within {B_j}, and charge trapping by a. For off-resonance coupling, mechanism i prevails with the charge separation rate and yield exhibiting an exponential dependence exp(−βR) on the d-a distance (R). Resonance coupling results in mechanism ii with the charge separation lifetime τ N^η and yield Y (1 + N^η)⁻¹ exhibiting a weak (algebraic) N and distance dependence. The power parameter η is determined by charge hopping random walk. Energetic control of the charge migration mechanism is exerted by the energetics of the ion pair state dB₁^±B₂… B_Na relative to the electronically excited donor doorway state d*B₁B₂… B_Na. The realization of charge separation via superexchange or hopping is determined by the base sequence within the bridge. Our energetic–dynamic relations, in conjunction with the energetic data for d*/d⁻ and for B/B⁺, determine the realization of the two distinct mechanisms in different hole donor systems, establishing the conditions for “chemistry at a distance” after charge transport in DNA. The energetic control of the charge migration mechanisms attained by the sequence specificity of the bridge is universal for large molecular-scale systems, for proteins, and for DNA.     

NMR evidence of a sharp change in a measure of local order in deeply supercooled confined water

Using NMR, we measure the proton chemical shift δ, of supercooled nanoconfined water in the temperature range 195 K < T < 350 K. Because δ is directly connected to the magnetic shielding tensor, we discuss the data in terms of the local hydrogen bond geometry and order. We argue that the derivative −( ln δ/T)_P should behave roughly as the constant pressure specific heat C_P(T), and we confirm this argument by detailed comparisons with literature values of C_P(T) in the range 290–370 K. We find that −( ln δ/T)_P displays a pronounced maximum upon crossing the locus of maximum correlation length at ≈240 K, consistent with the liquid-liquid critical point hypothesis for water, which predicts that C_P(T) displays a maximum on crossing the Widom line.     

A 55-kDa protein isolated from human cells shows DNA glycosylase activity toward 3,N4-ethenocytosine and the G/T mismatch

Etheno adducts in DNA arise from multiple endogenous and exogenous sources. Of these adducts we have reported that, 1,N⁶-ethenoadenine (A) and 3,N⁴-ethenocytosine (C) are removed from DNA by two separate DNA glycosylases. We later confirmed these results by using a gene knockout mouse lacking alkylpurine-DNA-N-glycosylase, which excises A. The present work is directed toward identifying and purifying the human glycosylase activity releasing C. HeLa cells were subjected to multiple steps of column chromatography, including two C-DNA affinity columns, which resulted in >1,000-fold purification. Isolation and renaturation of the protein from SDS/polyacrylamide gel showed that the C activity resides in a 55-kDa polypeptide. This apparent molecular mass is approximately the same as reported for the human G/T mismatch thymine-DNA glycosylase. This latter activity copurified to the final column step and was present in the isolated protein band having C-DNA glycosylase activity. In addition, oligonucleotides containing CG or G/T(U), could compete for C protein binding, further indicating that the C-DNA glycosylase is specific for both types of substrates in recognition. The same substrate specificity for C also was observed in a recombinant G/T mismatch DNA glycosylase from the thermophilic bacterium, Methanobacterium thermoautotrophicum THF.     

ER-27319, an acridone-related compound, inhibits release of antigen-induced allergic mediators from mast cells by selective inhibition of Fcɛ receptor I-mediated activation of Syk

Engagement of the mast cell high-affinity receptor for immunoglobulin E (IgE), FcRI, induces tyrosine phosphorylation of Syk, a non-receptor tyrosine kinase, that has been demonstrated as critical for degranulation. Herein we describe a synthetic compound, ER-27319, as a potent and selective inhibitor of antigen or anti-IgE-mediated degranulation of rodent and human mast cells. ER-27319 affected neither Lyn kinase activity nor the antigen-induced phosphorylation of the FcRI but did effectively inhibit the tyrosine phosphorylation of Syk and thus its activity. As a consequence, tyrosine phosphorylation of phospholipase C-γ1, generation of inositol phosphates, release of arachidonic acid, and secretion of histamine and tumor necrosis factor α were also inhibited. ER-27319 did not inhibit the anti-CD3-induced tyrosine phosphorylation of phospholipase C-γ1 in Jurkat T cells, demonstrating a specificity for Syk-induced signals. In contrast the tyrosine phosphorylation and activation of Syk, induced by in vitro incubation with the phosphorylated immunoreceptor tyrosine-based activation motif (ITAM) of FcRI γ subunit or by antigen activation of RBL-2H3 cells, was specifically inhibited by ER-27319. However, when ER-27319 was added to immunoprecipitated Syk, derived from activated cells, no effect was seen on Syk activity. ER-27319 did not inhibit the tyrosine phosphorylation of Syk induced by activation in the presence of Igβ ITAM or the anti-IgM-induced phosphorylation of Syk in human peripheral B cells. Therefore, ER-27319 selectively interferes with the FcRI γ phospho-ITAM activation of Syk in vitro and in intact cells. These results confirm the importance of Syk in FcRI-mediated responses in mast cells and demonstrate the mast cell selectivity and therapeutic potential of ER-27319 in the treatment of allergic disease.     

Acetylcholine receptor ɛ-subunit deletion causes muscle weakness and atrophy in juvenile and adult mice

In mammalian muscle a postnatal switch in functional properties of neuromuscular transmission occurs when miniature end plate currents become shorter and the conductance and Ca²⁺ permeability of end plate channels increases. These changes are due to replacement during early neonatal development of the γ-subunit of the fetal acetylcholine receptor (AChR) by the -subunit. The long-term functional consequences of this switch for neuromuscular transmission and motor behavior of the animal remained elusive. We report that deletion of the -subunit gene caused in homozygous mutant mice the persistence of γ-subunit gene expression in juvenile and adult animals. Neuromuscular transmission in these animals is based on fetal type AChRs present in the end plate at reduced density. Impaired neuromuscular transmission, progressive muscle weakness, and atrophy caused premature death 2 to 3 months after birth. The results demonstrate that postnatal incorporation into the end plate of -subunit containing AChRs is essential for normal development of skeletal muscle.     

Targeted deletion of alkylpurine-DNA-N-glycosylase in mice eliminates repair of 1,N6-ethenoadenine and hypoxanthine but not of 3,N4-ethenocytosine or 8-oxoguanine

It has previously been reported that 1,N⁶-ethenoadenine (A), deaminated adenine (hypoxanthine, Hx), and 7,8-dihydro-8-oxoguanine (8-oxoG), but not 3,N⁴-ethenocytosine (C), are released from DNA in vitro by the DNA repair enzyme alkylpurine-DNA-N-glycosylase (APNG). To assess the potential contribution of APNG to the repair of each of these mutagenic lesions in vivo, we have used cell-free extracts of tissues from APNG-null mutant mice and wild-type controls. The ability of these extracts to cleave defined oligomers containing a single modified base was determined. The results showed that both testes and liver cells of these knockout mice completely lacked activity toward oligonucleotides containing A and Hx, but retained wild-type levels of activity for C and 8-oxoG. These findings indicate that (i) the previously identified A-DNA glycosylase and Hx-DNA glycosylase activities are functions of APNG; (ii) the two structurally closely related mutagenic adducts A and C are repaired by separate gene products; and (iii) APNG does not contribute detectably to the repair of 8-oxoG.     

Slow and fast dietary proteins differently modulate postprandial protein accretion

The speed of absorption of dietary amino acids by the gut varies according to the type of ingested dietary protein. This could affect postprandial protein synthesis, breakdown, and deposition. To test this hypothesis, two intrinsically ¹³C-leucine-labeled milk proteins, casein (CAS) and whey protein (WP), of different physicochemical properties were ingested as one single meal by healthy adults. Postprandial whole body leucine kinetics were assessed by using a dual tracer methodology. WP induced a dramatic but short increase of plasma amino acids. CAS induced a prolonged plateau of moderate hyperaminoacidemia, probably because of a slow gastric emptying. Whole body protein breakdown was inhibited by 34% after CAS ingestion but not after WP ingestion. Postprandial protein synthesis was stimulated by 68% with the WP meal and to a lesser extent (+31%) with the CAS meal. Postprandial whole body leucine oxidation over 7 h was lower with CAS (272 ± 91 μmolkg⁻¹) than with WP (373 ± 56 μmolkg⁻¹). Leucine intake was identical in both meals (380 μmolkg⁻¹). Therefore, net leucine balance over the 7 h after the meal was more positive with CAS than with WP (P < 0.05, WP vs. CAS). In conclusion, the speed of protein digestion and amino acid absorption from the gut has a major effect on whole body protein anabolism after one single meal. By analogy with carbohydrate metabolism, slow and fast proteins modulate the postprandial metabolic response, a concept to be applied to wasting situations.     

Reexamination of the mechanism of hydroxyl radical adducts formed from the reaction between familial amyotrophic lateral sclerosis-associated Cu,Zn superoxide dismutase mutants and H2O2

Amyotrophic lateral sclerosis (ALS) involves the progressive degeneration of motor neurons in the spinal cord and motor cortex. Mutations to Cu,Zn superoxide dismutase (SOD) linked with familial ALS are reported to increase hydroxyl radical adduct formation from hydrogen peroxide as measured by spin trapping with 5,5′-dimethyl-1-pyrrolline N-oxide (DMPO). In the present study, we have used oxygen-17-enriched water and H₂O₂ to reinvestigate the mechanism of DMPO/OH formation from the SOD and SOD mutants. The relative ratios of DMPO/¹⁷OH and DMPO/¹⁶OH formed in the Fenton reaction were 90% and 10%, respectively, reflecting the ratios of H₂¹⁷O₂ to H₂¹⁶O₂. The reaction of the WT SOD with H₂¹⁷O₂ in bicarbonate/CO₂ buffer yielded 63% DMPO/¹⁷OH and 37% DMPO/¹⁶OH. Similar results were obtained from the reaction between familial ALS SOD mutants and H₂¹⁷O₂: DMPO/¹⁷OH (64%); DMPO/¹⁶OH (36%) from A4V and DMPO/¹⁷OH (62%); and DMPO/¹⁶OH (38%) from G93A. These results were confirmed further by using 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide spin trap, a phosphorylated analog of DMPO. Contrary to earlier reports, the present results indicate that a significant fraction of DMPO/OH formed during the reaction of SOD and familial ALS SOD mutants with H₂O₂ is derived from the incorporation of oxygen from water due to oxidation of DMPO to DMPO/OH presumably via DMPO radical cation. No differences were detected between WT and mutant SODs, neither in the concentration of DMPO/OH or DEPMPO/OH formed nor in the relative incorporation of oxygen from H₂O₂ or water.     

Dynamic electron microscopy of ATP-induced myosin head movement in living muscle thick filaments

Although muscle contraction is known to result from movement of the myosin heads on the thick filaments while attached to the thin filaments, the myosin head movement coupled with ATP hydrolysis still remains to be investigated. Using a gas environmental (hydration) chamber, in which biological specimens can be kept in wet state, we succeeded in recording images of living muscle thick filaments with gold position markers attached to the myosin heads. The position of individual myosin heads did not change appreciably with time in the absence of ATP, indicating stability of the myosin head mean position. On application of ATP, the position of individual myosin heads was found to move by ≈20 nm along the filament axis, whereas no appreciable movement of the filaments was detected. The ATP-induced myosin head movement was not observed in filaments in which ATPase activity of the myosin heads was eliminated. Application of ADP produced no appreciable myosin head movement. These results show that the ATP-induced myosin head movement takes place in the absence of the thin filaments. Because ATP reacts rapidly with the myosin head (M) to form the complex (MADPP_i) with an average lifetime of >10 s, the observed myosin head movement may be mostly associated with reaction, M + ATP → MADPP_i. This work will open a new research field to study dynamic structural changes of individual biomolecules, which are kept in a living state in an electron microscope.     

Characters of irreducible Harish-Chandra modules and Goldie ranks of their annihilators

Assume that G_C is a simply connected complex semi-simple Lie group with Lie algebra [unk]. Let G G_C be a real form, H G be a maximally split Cartan subgroup with Lie algebra [unk]₀, [unk] = [unk]₀ [unk] C, and P [unk]^* be the weight lattice. If X is an irreducible Harish-Chandra module with infinitesimal character λ [unk]^*, one can associate to X a family {θ(μ): μ λ + P} of Z-linear combinations of distribution characters of G, so that θ(λ) = X. θ(μ) is irreducible when μ lies in C_λ, a certain positive “Weyl” chamber containing λ. In this case let Ann θ(μ) be its annihilator in U([unk]) and set p(μ) = Goldie rank of U([unk])/ Ann θ(μ). Let d = Gelfand-Kirillov dimension of X. For most x [unk]₀ if exp tx is regular for small t > 0 then (i) c(μ) = lim_t→0⁺t^d θ(μ) (exp tx) exists for all μ λ + P; (ii) c(μ) extends to a homogeneous Weyl group harmonic polynomial on [unk]^* of degree ½(dim G - dim H) - d; (iii) up to a constant, c = the polynomial extending p to [unk]^*. c is said to be the character polynomial of Ann X.