Structural and ligand recognition characteristics of an acetylcholine-binding protein from Aplysia californica

We generated an acetylcholine-binding protein from Aplysia californica by synthesis of a cDNA found in existing data bases and its expression in mammalian cell culture. Its subunit assembly and ligand recognition behavior were compared with the binding protein previously derived from Lymnaea stagnalis. The secreted proteins were purified by elution from columns of attached antibodies directed to the FLAG epitope encoded in the expression construct. Although the sequences of the two proteins from marine and fresh water mollusks exhibit the characteristic features of the extracellular domain of the nicotinic receptor, they only possess 33% amino acid identity. Both assemble as stable pentamers with five binding sites per pentamer, yet they show distinguishing features of stability and sensitivity to epitope tag placement. Both proteins exhibit changes in tryptophan fluorescence upon ligand binding; however, the magnitude of the changes differs greatly. Moreover, certain ligands show marked differences in dissociation constants for the two proteins and can be regarded as distinguishing or signature ligands. Hence, the two soluble proteins from mollusks, which can be studied by a variety of physical methods, become discrete surrogate proteins for the extracellular domains of distinct subtypes of nicotinic acetylcholine receptors.     

Purification and characterization of enzymes exhibiting beta-D-xylosidase activities in stem tissues of Arabidopsis

This work describes the purification and characterization of enzymes that exhibit beta-d-xylosidase activity in stem tissues of Arabidopsis. This is the first detailed investigation that concerns the characterization of catalytic properties and sequence identity of enzymes with beta-D-xylosidase activities in a dicotyledonous plant. Three different enzymes, ARAf, XYL4, and XYL1 with apparent molecular masses of 75, 67, and 64 kD, respectively, were purified to homogeneity. ARAf was identified as a putative alpha-L-arabinofuranosidase, and XYL4 and XYL1 as putative beta-D-xylosidases using matrix-assisted laser-desorption ionization time of flight. ARAf belongs to family 51 and XYL4 and XYL1 to family 3 of glycoside hydrolases. ARAf and XYL1 have highest specificity for p-nitrophenyl-alpha-L-arabinofuranoside and XYL4 for p-nitrophenyl-beta-D-xylopyranoside and natural substrates such as xylobiose and xylotetraose. XYL4 was shown to release mainly D-Xyl from oat spelt xylan, rye arabinoxylan, wheat arabinoxylan, and oligoarabinoxylans. ARAf and XYL1 can also release D-Xyl from these substrates but less efficiently than XYL4. Moreover, they can also release L-Ara from arabinoxylans and arabinan. Overall, the results indicate that XYL4 possesses enzymatic specificity characteristic for a beta-D-xylosidase, while ARAf and XYL1 act as bifunctional alpha-L-arabinofuranosidase/beta-D-xylosidases. Analysis of the activity of these three enzymes in stem tissues at different stages of development has shown that young stems possess the highest activities for all three enzymes in comparison to the activities of the enzymes present in stems at older stages of development. High enzyme activities are most likely related to the necessary modifications of cell wall structure occurring during plant growth.     

The influence of detergents and active components of detergent on bioproduction of organic matters and enzymatic activity of some species of fungi.

Detergent (Merix, "Merima " Krusevac) applied in concentration of 1% vol. showed specific influence on the bioproduction of some 15 different amino acids and on the enzyme activity of the species of fungi A. niger, A. alternata and T. roseum. Detergent has significantly stimulated the production of 15 analyzed amino acids of the fungi species A. niger. The same applied concentration of detergent has decreased or considerably decreased the production of some 14 of totally 15 analyzed amino acids of investigated fungi species A. alternata and T. roseum. The enzyme activity of the fungi A. niger was more intensive in relation to the species A. alternata and T. roseum during the experimental period or in some phases of the experimental period. The detergent component, ethoxyled oleyl-cetyl alcohol, in concentration of 0.01%, 0.1% and 1% showed an inhibitory effect, or significant inhibitory effect on the enzyme activity of the examined species of fungi (A. niger, A. alternata and T. roseum).     

Efficiency of polylactide/polyglycolide copolymers bone replacements in bone defects healing measured by densitometry

Healing of bone defects is the most frequent cause of failure in surgical treatments of an odontogenic ostitic processes. The aim of this study was to determine successfulness in healing of bone defects after implantation of alloplastic co-polymer-polyglycol bone implant. A group of 45 cases with periradicular ostitic processes were examined. The densitometric measurements were taken from radiovisiographic dental x-ray images. Patients were observed throughout a period of one year. The results obtained were analyzed and presented graphically. Thirty-eight patients (84%) were treated successfully, and seven patients (16%) showed unsuccessful healing of the bone defects. The results obtained indicate that polyglycol copolymer bone implants can be successfully used in a treatment of odontogenically caused bone defects. Their fundamental advantage is a slow biodegradation, which ensures a more suitable area for the apposition of a new bone in the defect, simple application in clinical work and the possibility of a mutual combination of all three available forms.     

Prevention of cardiac hypertrophy and heart failure by silencing of NF-kappaB

Activation of the nuclear factor (NF)-κB signaling pathway may be associated with the development of cardiac hypertrophy and its transition to heart failure (HF). The transgenic Myo-Tg mouse develops hypertrophy and HF as a result of overexpression of myotrophin in the heart associated with an elevated level of NF-κB activity. Using this mouse model and an NF-κB-targeted gene array, we first determined the components of NF-κB signaling cascade and the NF-κB-linked genes that are expressed during the progression to cardiac hypertrophy and HF. Second, we explored the effects of inhibition of NF-κB signaling events by using a gene knockdown approach: RNA interference through delivery of a short hairpin RNA against NF-κB p65 using a lentiviral vector (L-sh-p65). When the short hairpin RNA was delivered directly into the hearts of 10-week-old Myo-Tg mice, there was a significant regression of cardiac hypertrophy, associated with a significant reduction in NF-κB activation and atrial natriuretic factor expression. Our data suggest, for the first time, that inhibition of NF-κB using direct gene delivery of sh-p65 RNA results in regression of cardiac hypertrophy. These data validate NF-κB as a therapeutic target to prevent hypertrophy/HF.     

Purification, functional characterization, cloning, and identification of mutants of a seed-specific arabinan hydrolase in Arabidopsis

This work describes the purification and characterization of an enzyme that exhibits arabinan hydrolase activity in seeds of Arabidopsis thaliana. The enzyme, designated XYL3, had an apparent molecular mass of 80 kDa when purified to homogeneity, and was identified using MALDI-TOF (matrix-assisted laser desorption ionization-time of flight) as a putative beta-D-xylosidase that belongs to family 3 of glycoside hydrolases encoded by gene At5g09730. XYL3 hydrolysed synthetic substrates such as p-nitrophenyl-alpha-L-arabinofuranoside and p-nitrophenyl-beta-D-xyloside with similar catalytic efficiency. XYL3 released L-arabinose from (1-->5)-alpha-L-arabinofuranobiose, arabinoxylan, sugar beet arabinan, and debranched arabinan. The enzyme hydrolysed both arabinosyl-substituted side group residues and terminal arabinofuranosyl residues (1-->5)-alpha-linked to the arabinan backbone. This indicates that XYL3 is able to degrade all terminal arabinosyl residues and suggests that it participates in the in-vivo hydrolysis of arabinan. Analysis of gene expression patterns by semi-quantitative RT-PCR, in-situ hybridization and a promoter-GUS fusion demonstrated that AtBX3 was specifically expressed in the seed endosperm at the globular stage of the embryo. Immunolocalization using LM6 anti-arabinan antisera found that arabinan, the XYL3 substrate, was also present in this seed tissue. T-DNA null mutants for AtBX3 were identified. The mutant plants lacked the alpha-L-arabinofuranosidase and beta-D-xylosidase activities corresponding to XYL3. Mutants showed reduced seed size and are delayed in seedling germination compared with the wild type.     

Endogenous vascular remodeling in ischemic skeletal muscle: a role for nitric oxide.

To test the hypothesis that nitric oxide (NO) production is essential for endogenous vascular remodeling in ischemic skeletal muscle, 22 New Zealand White rabbits were chronically instrumented with transit-time flow probes on the common iliac arteries and underwent femoral ligation to produce unilateral hindlimb ischemia. Iliac blood flow and arterial pressure were recorded at rest and during a graded exercise test. An osmotic pump connected to a femoral arterial catheter continuously delivered N-nitro-l-arginine methyl ester (a NO synthase inhibitor) or a control solution (N-nitro-d-arginine methyl ester or phenylephrine) to the ischemic limb over a 2-wk period. At 1, 3, and 6 wk after femoral ligation, maximal treadmill exercise blood flow in the ischemic limb was reduced compared with baseline in each group. However, maximal exercise blood flow was significantly (P < 0.05) lower in the l-NAME-treated group than in controls for the duration of the study: 48 +/- 4 vs. 60 +/- 5 ml/min at 6 wk. Consistent with the reduction in maximal blood flow response, the duration of voluntary exercise was also substantially (P < 0.05) shorter in the l-NAME-treated group: 539 +/- 67 vs. 889 +/- 87 s. Resting blood flow was unaffected by femoral ligation in either group. The results of this study show that endogenous vascular remodeling, which partially alleviated the initial deficit in blood flow, was interrupted by NO synthase inhibition. Therefore, we conclude that NO is essential for endogenous collateral development and angiogenesis in ischemic skeletal muscle in the rabbit.     

Muscle pump does not enhance blood flow in exercising skeletal muscle.

The muscle pump theory holds that contraction aids muscle perfusion by emptying the venous circulation, which lowers venous pressure during relaxation and increases the pressure gradient across the muscle. We reasoned that the influence of a reduction in venous pressure could be determined after maximal pharmacological vasodilation, in which the changes in vascular tone would be minimized. Mongrel dogs (n = 7), instrumented for measurement of hindlimb blood flow, ran on a treadmill during continuous intra-arterial infusion of saline or adenosine (15-35 mg/min). Adenosine infusion was initiated at rest to achieve the highest blood flow possible. Peak hindlimb blood flow during exercise increased from baseline by 438 +/- 34 ml/min under saline conditions but decreased by 27 +/- 18 ml/min during adenosine infusion. The absence of an increase in blood flow in the vasodilated limb indicates that any change in venous pressure elicited by the muscle pump was not adequate to elevate hindlimb blood flow. The implication of this finding is that the hyperemic response to exercise is primarily attributable to vasodilation in the skeletal muscle vasculature.     

Sympathetic restraint of muscle blood flow at the onset of dynamic exercise.

Little attention has focused on sympathetic influences on skeletal muscle blood flow at the onset of exercise. We hypothesized that 1) the sympathetic nervous system constrains muscle blood flow and 2) the decline from peak blood flow is mediated by increasing sympathetic vasoconstrictor tone. Mongrel dogs (n = 7) ran on a treadmill after intra-arterial infusion of saline (control) or combined alpha(1)- and alpha(2)-adrenergic blockade (prazosin and rauwolscine). Immediate and rapid increases in hindlimb blood flow occurred at commencement of exercise with peak iliac blood flows averaging 933 +/- 79 and 1,227 +/- 90 ml/min during control and blockade conditions, respectively. At 1 min of exercise, hindlimb blood flow had decreased to 629 +/- 54 and 1,057 +/- 89 ml/min. In the absence of sympathetic vasoconstrictor tone, there was an enhanced peak blood flow at the onset of exercise. In addition, alpha-blockade attenuated the overshoot of hindlimb blood flow compared with the control condition. These data suggest that an immediate and sustained increase in sympathetic outflow restrains hindlimb blood flow at the onset of exercise and is responsible, at least in part, for an overshoot of blood flow to exercising skeletal muscle.