Active axial stress in mouse aorta

The study verifies the development of active axial stress in the wall of mouse aorta over a range of physiological loads when the smooth muscle cells are stimulated to contract. The results obtained show that the active axial stress is virtually independent of the magnitude of pressure, but depends predominately on the longitudinal stretch ratio. The dependence is non-monotonic and is similar to the active stress-stretch dependence in the circumferential direction reported in the literature. The expression for the active axial stress fitted to the experimental data shows that the maximum active stress is developed at longitudinal stretch ratio 1.81, and 1.56 is the longitudinal stretch ratio below which the stimulation does not generate active stress. The study shows that the magnitude of active axial stress is smaller than the active circumferential stress. There is need for more experimental investigations on the active response of different types of arteries from different species and pathological conditions. The results of these studies can promote building of refined constrictive models in vascular rheology.     

Series-type enzyme deactivations: Influence of intermediate activity on deactivation kinetics

A series-type enzyme deactivation model involving an active enzyme precursor is proposed wherein the enzyme activity is a weighted function of the active enzyme states. The active enzyme precursor may be less active, as active or more active than the initial enzyme form. The proposed model is shown to fit the soluble and immobilized enzyme deactivation data presented reasonably well. Some enzymes exhibit a ‘compensation-like’ effect. In other enzymes, if the deactivation rate coefficient for the second step, k₂, is zero, then the activity may stabilize to a value that depends upon the relative activities of the two active enzyme states.     

A kinetic model for determining the consequences of electrogenic active transport in cardiac muscle

When active transport is electrogenic in a tissue that is continuously active, such as cardiac muscle, the active transport current is as important in the generation of the action potential as are the passive currents. A thermodynamically constrained kinetic model of electrogenic active transport of sodium and potassium ions has been developed in which the influences of voltage and chemical composition are explicitly defined. This model is coupled to a system of passive permeabilities, of the minimum degree of complexity, to simulate the integrated activity of active and passive ion transport in the generation of the cardiac action potential. Results of preliminary simulations indicate that electrogenic active transport provides a mechanism for slowly changing currents both within the time scale of an action potential as well as of many action potentials. The presence of active transport also complicates the interpretation of isotopic flux measurements and the separation of currents.     

Oxidation of a single active site suffices for the functional inactivation of the dimeric Bacillus subtilis OhrR repressor in vitro

Bacillus subtilis OhrR is a dimeric repressor that senses organic peroxides and regulates the expression of the OhrA peroxiredoxin. Derepression results from oxidation of an active site cysteine which ultimately results in formation of a mixed disulfide with a low molecular weight thiol, a cyclic sulfenamide, or overoxidation to the sulfinic or sulfonic acids. We expressed a single-chain OhrR (scOhrR) in which the two monomers were connected by a short amino-acid linker. scOhrR variants containing only one active site cysteine were fully functional as repressors and still responded, albeit with reduced efficacy, to organic peroxides in vivo. Biochemical analyses indicate that oxidation at a single active site is sufficient for derepression regardless of the fate of the active site cysteine. scOhrR with only one active site cysteine in the amino-terminal domain is inactivated at rates comparable to wild-type whereas when the active site is in the carboxyl-terminal domain the protein is inactivated much more slowly. The incomplete derepression noted for single active site variants of scOhrR in vivo is consistent with the hypothesis that protein reduction regenerates active repressor and that, in the cell, oxidation of the second active site may also contribute to derepression.     

Regeneration of the active zone at the frog neuromuscular junction

The active zone is a unique specialization of the presynaptic membrane and is believed to be the site of transmitter release. The formation of the active zone and the relationship of this process to transmitter release were studied at reinnervated neuromuscular junctions in the frog. At different times after a nerve crush, the cutaneous pectoris muscles were examined with intracellular recording recording and freeze-fracture electron microscopy. The P face of a normal active zone typically consists of two double rows of particles lined up in a continuous segment located opposite a junctional fold. In the initial stage of reinnervation, clusters of large intramembrane particles surrounding membrane elevations appeared on the P face of nerve terminals. Like normal active zones, these clusters were aligned with junctional folds. Vesicle openings, which indicate transmitter release, were seen at these primitive active zones, even though intramembrane particles were not yet organized into the normal pattern of two double rows. The length of active zones at this stage was only approximately 15% of normal. During the secondary stage, every junction was reinnervated and most active zones had begun to organize into the normal pattern with normal orientation. Unlike normal, there were often two or more discontinuous short segments of active zone aligned with the same junctional fold. The total length of active zone per junctional fold increased to one-third of normal, mainly because of the greater number of segments. In the third stage, the number of active zone segments per junctional fold showed almost no change when compared with the secondary stage. However, individual segments elongated and increased the total length of all active zone segments per junctional fold to about two-thirds of the normal length. The dynamic process culminated in the final stage, during which elongating active zones appeared to join together and the number of active zone segments per junctional fold decreased to normal. Thus, in most regions, regeneration of the active zones was complete. These results suggest that the normal organization of two double rows is not necessary for the active zone to be functional. Furthermore, localization of regenerating active zones is related to junctional folds and/or their associated structures.     

Preferential localization of glutamate receptors opposite sites of high presynaptic release

BACKGROUND: The localization of glutamate receptors is essential for the formation and plasticity of excitatory synapses. These receptors cluster opposite neurotransmitter release sites of glutamatergic neurons, but these release sites have heterogeneous structural and functional properties. At the Drosophila neuromuscular junction, receptors expressed in a single postsynaptic cell are confronted with an array of hundreds of apposed active zones. Hence, this is an ideal preparation for the investigation of whether receptor clustering is sensitive to the morphological and physiological properties of the apposed active zones. RESULTS: To investigate the relationship between the localization of glutamate receptors and the properties of the apposed active zones, we investigated receptor localization in mutants in which receptors are limited. We find that receptors are not uniformly distributed opposite the full array of active zones but that some active zones have a disproportionately large share of receptors as assayed by receptor levels and response to transmitter. The active zones at which receptors preferentially cluster are larger and have a higher neurotransmitter release probability than the average active zone. We find a similar relationship between glutamate receptor clusters and active-zone size at wild-type synapses. CONCLUSIONS: When confronted with an array of active zones, glutamate receptors preferentially cluster opposite the largest and most physiologically active sites. These results suggest an activity-dependent matching of pre- and postsynaptic function at the level of a single active zone.     

Nuclease sensitivity of active chromatin.

The active regions of chicken erythrocyte nuclei were labeled using the standard DNase I directed nick translation reaction. These nuclei were then used to study the characteristics and, in particular, the nuclease sensitivity of active genes. Although DNase I specifically attacks active genes, micrococcal nuclease solubilizes these regions to about the same degree as the total DNA. On the other hand micrococcal nuclease does selectively cut the internucleosomal regions of active genes resulting in the appearance of mononucleosomal fraction which is enriched in active gene DNA. A small percentage of the active chromatin is also released from the nucleus by low speed centrifugation following micrococcal nuclease treatment. The factors which make active genes sensitive to DNase I were shown to reside on individual nucleosomes from these regions. This was established by showing that isolated active mononucleosomes were preferentially sensitive to DNase I digestion. Although the high mobility group proteins are essential for the maintenance of DNase I sensitivity in active regions, these proteins are not necessary for the formation of the conformation which makes these genes preferentially accessible to micrococcal nuclease. The techniques employed in this paper enable one to study the chromatin structure of the entire population of actively expressed genes. Previous studies have elucidated the structure of a few special highly prevalent genes such as ovalbumin and hemoglobin. The results of this paper show that this special conformation is a general feature of all active genes irregardless of the extent of expression.     

The Effect of Shortening on the Time-Course of Active State Decay

The active state describes the force developed in a muscle when the contractile elements are neither lengthening nor shortening. Recently it was suggested that perturbations used to measure the active state also alter the time-course of the active state. The present research was undertaken to assess quantitatively the effect of two such perturbations, isotonic shortening and quick release, on the active state in frog sartorius muscle. Methods were developed which allowed the determination of active state points following periods of controlled isotonic shortening or quick release early in the contraction cycle. All experiments were carried out within the plateau region of the length-tension curve. Both isotonic shortening and quick release altered the active state decay. The active state force decreased as the extent of shortening or release was increased. For each 0.1 mm of isotonic shortening there was a 2% decrease in active state force. Quick release produced a larger decrement. From this data we conclude that the time-course of active state can be measured only in relative terms because it is altered by the motion which takes place in the contractile machine while the active state is being measured. This finding helps to resolve paradoxes in the literature relating to the time-course of the active state, calculated and experimentally determined isometric tetanic myograms, and the heat of shortening.     

变性过程中铜锌超氧化物歧化酶的活性通道

在酶的盐酸胍变性和热变性过程中,尝试采用电荷传递反应分析方法和电子自旋共振方法考察了酶活性部位的构象变化。酶活力与构象的变化行为表明,酶的活性部位通道先于酶分子的整体构象而发生变化,它是与酶的失活同时发生的。尽管酶活性部位中的金属离子保证了酶较高的稳定性,但酶的活性部位,特别是活性通道仍然是相对脆弱的。     

Use of pseudosubstrate affinity to measure active protein kinase A

Traditional cAMP-dependent protein kinase (also known as protein kinase A [PKA]) assays, which are based on substrate phosphorylation, often have high background activity from other kinases, thereby limiting sensitivity and making it difficult to detect low levels of active PKA in cell lysates. Therefore, a better technique that measures active PKA in crude cell lysates undoubtedly is necessary. We developed an efficient and sensitive assay to compare active PKA levels based on binding of the active PKA catalytic subunit to its pseudosubstrate domain inhibitor (PKI) fused with glutathione S-transferase (GST-PKI). This pseudosubstrate affinity assay can detect variations in the active PKA levels in the presence of common inducers of PKA activity such as forskolin and prostaglandins. It has resolution to detect a concentration-dependent curve of active PKA in a linear range, and it also has sensitivity to detect up to 2.5 ng of active enzyme. An observed change in the binding affinity between PKA and PKI in the presence of the PKA inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H89) shows that this assay can be successfully used to measure how active PKA is affected by specific inhibitors. We conclude that this method is a simple, inexpensive, and nonhazardous method to compare active PKA levels with high sensitivity and specificity with negligible background.     

A comparison of the phosphorylated and unphosphorylated forms of isocitrate dehydrogenase from Escherichia coli ML308

NADP+ can protect active isocitrate dehydrogenase against attack by several proteases. Inactive phosphorylated isocitrate dehydrogenase is much less susceptible to proteolysis than the active enzyme, and it is not protected by NADP+. The results suggest that binding of NADP+ to, or phosphorylation of, active isocitrate dehydrogenase induces similar conformational states. Fluorescence titration experiments show that NADPH can bind to active but not to inactive isocitrate dehydrogenase. It is suggested that the phosphorylation of isocitrate dehydrogenase may occur close to its coenzyme binding site.     

HslVU ATP-dependent Protease Utilizes Maximally Six among Twelve Threonine Active Sites during Proteolysis

HslVU is a bacterial ATP-dependent protease distantly related to eukaryotic proteasomes consisting of hexameric HslU ATPase and dodecameric HslV protease. As a homolog of the 20 S proteasome β-subunits, HslV also uses the N-terminal threonine as the active site residue. However, unlike the proteasome that has only 6 active sites among the 14 β-subunits, HslV has 12 active sites that could potentially contribute to proteolytic activity. Here, by using a series of HslV dodecamers containing different numbers of active sites, we demonstrate that like the proteasome, HslV with only ∼6 active sites is sufficient to support full catalytic activity. However, a further reduction of the number of active sites leads to a proportional decrease in activity. Using proteasome inhibitors, we also demonstrate that substrate-mediated stabilization of the HslV-HslU interaction remains unchanged until the number of the active sites is decreased to ∼6 but is gradually compromised upon further reduction. These results with a mathematical model suggest HslVU utilizes no more than 6 active sites at any given time, presumably because of the action of HslU. These results also suggest that each ATP-bound HslU subunit activates one HslV subunit and that substrate bound to the HslV active site stimulates the HslU ATPase activity by stabilizing the HslV-HslU interaction. We propose this mechanism plays an important role in supporting complete degradation of substrates while preventing wasteful ATP hydrolysis in the resting state by controlling the interaction between HslV and HslU through the catalytic engagement of the proteolytic active sites.     

Peptide binding to active class II MHC protein on the cell surface

Solution studies have demonstrated the existence of two functionally distinct isomers of empty class II MHC: an active isomer that binds peptide and an inactive isomer that does not. Empty MHC molecules on the surface of APCs can load antigenic peptides directly from the extracellular medium, facilitating the generation of a diverse peptide repertoire for T cell presentation. In this report, we examine I-Ek on the surface of Chinese hamster ovary cells with respect to the active and inactive isomers. As in the case of purified soluble active I-Ek, active I-Ek on the cell surface is unstable, decaying to the inactive form in approximately 14 min. Evidence is presented suggesting that at steady state <1% of the total cell surface I-Ek is active and that a significant fraction of these active molecules originates from intracellular pools as well as reactivation of inactive cell surface I-EK.     

Muscle kinematics for minimal work of breathing.

A mathematical model was analyzed to obtain a quantitative and testable representation of the long-standing hypothesis that the respiratory muscles drive the chest wall along the trajectory for which the work of breathing is minimal. The respiratory system was modeled as a linear elastic system that can be expanded either by pressure applied at the airway opening (passive inflation) or by active forces in respiratory muscles (active inflation). The work of active expansion was calculated, and the distribution of muscle forces that produces a given lung expansion with minimal work was computed. The calculated expression for muscle force is complicated, but the corresponding kinematics of muscle shortening is simple: active inspiratory muscles shorten more during active inflation than during passive inflation, and the ratio of active to passive shortening is the same for all active muscles. In addition, the ratio of the minimal work done by respiratory muscles during active inflation to work required for passive inflation is the same as the ratio of active to passive muscle shortening. The minimal-work hypothesis was tested by measurement of the passive and active shortening of the internal intercostal muscles in the parasternal region of two interspaces in five supine anesthetized dogs. Fractional changes in muscle length were measured by sonomicrometry during passive inflation, during quiet breathing, and during forceful inspiratory efforts against a closed airway. Active muscle shortening during quiet breathing was, on average, 70% greater than passive shortening, but it was only weakly correlated with passive shortening. Active shortening inferred from the data for more forceful inspiratory efforts was approximately 40% greater than passive shortening and was highly correlated with passive shortening. These data support the hypothesis that, during forceful inspiratory efforts, muscle activation is coordinated so as to expand the chest wall with minimal work.     

Deficient repair of the transcribed strand of active genes in Cockayne's syndrome cells.

Removal of ultraviolet light induced cyclobutane pyrimidine dimers (CPD) from active and inactive genes was analyzed in cells derived from patients suffering from the hereditary disease Cockayne's syndrome (CS) using strand specific probes. The results indicate that the defect in CS cells affects two levels of repair of lesions in active genes. Firstly, CS cells are deficient in selective repair of the transcribed strand of active genes. In these cells the rate and efficiency of repair of CPD are equal for the transcribed and the nontranscribed strand of the active ADA and DHFR genes. In normal cells on the other hand, the transcribed strand of these genes is repaired faster than the nontranscribed strand. However, the nontranscribed strand is still repaired more efficiently than the inactive 754 gene and the gene coding for coagulation factor IX. Secondly, the repair level of active genes in CS cells exceeds that of inactive loci but is slower than the nontranscribed strand of active genes in normal cells. Our results support the model that CS cells lack a factor which is involved in targeting repair enzymes specifically towards DNA damage located in (potentially) active DNA.     

Partial purification and characterization of exudate gelatinase in the acute phase of carrageenin-induced inflammation in rats

Gelatinase has been partially purified from exudate in the acute phase of carrageenin-induced inflammation in rats. The enzyme occurs in a latent form that can be activated with 4-aminophenylmercuric acetate (APMA). The latent gelatinase was separated into an active gelatinase and a protein fraction by zinc-chelating Sepharose 6B column chromatography in the final step of purification, suggesting that the latent gelatinase is an enzyme-inhibitor complex. The pH optimum of the active gelatinase is about 7.5 and no reactivity toward native type I collagen or alpha-casein was detected. The molecular weights of the latent and active gelatinases were about 245,000 and about 185,000, respectively, as determined by gel filtration on Sephadex G-200. On the other hand, both latent and active gelatinases occurred in multiple forms in SDS-substrate polyacrylamide gel electrophoresis; the latent gelatinase showed two bands with molecular weights of 105,000 and 69,000, and two additional bands of 88,000 and 83,000 appeared when the latent gelatinase was activated with APMA, while the active gelatinase showed all four species. The active gelatinase was inhibited by metallo-proteinase inhibitors, but not by serine- or cysteine-proteinase inhibitors, suggesting that the exudate gelatinase is a metallo-proteinase. The active gelatinase was also inhibited by serum proteins such as albumin and gamma-globulin, suggesting that gelatinase does not remain in an active form in the inflammatory lesion, where the vascular permeability is increased.     

氨基酸混合液脱色条件的比较选择

以四种粉末状活性炭作为脱色剂,对猪血粉水解液进行脱色比较,结果表明：江西产的４号粉末状活性炭脱色效果最佳。本文还对４号脱色炭的脱色条件进行了摸索研究。