Recognizing very distant sequence relationships among proteins by family profile analysis.

Family profile analysis (FPA), described in this paper, compares all available homologous amino acid sequences of a target family with the profile of a probe family while conventional sequence profile analysis (Gribskov M, Lüthy R, Eisenberg D. Meth Enzymol 1990;183:146-159) considers only a single target sequence in comparison with the probe family. The increased input of sequence information in FPA expands the range for sequence-based recognition of structural relationships. In the FPA algorithm, Zscores of each of the target sequences, obtained from a probe profile search over all known amino acid sequences, are averaged and then compared with the scores for sequences of 100 reference families in the same probe family search. The resulting F-Zscore of the target family, expressed in "effective standard deviations" of the mean Zscores of the reference families, with value above a threshold of 3.5 indicates a statistically significant evolutionary relationship between the target and probe families. The sensitivity of FPA to sequence information was tested with several protein families where distant relationships have been verified from known tertiary protein architectures, which included vitamin B6-dependent enzymes, (beta/alpha)8-barrel proteins, beta-trefoil proteins, and globins. In comparison to other methods, FPA proved to be significantly more sensitive, finding numerous new homologies. The FPA technique is not only useful to test a suspected relationship between probe and target families but also identifies possible target families in profile searches over all known primary structures.     

Fibrinopeptide A induces C-reactive protein expression through the ROS-ERK1/2/p38-NF-κB signal pathway in the human umbilical vascular endothelial cells

Atherosclerosis is a chronic inflammatory disease of the arterial wall. Inflammation causes endothelial injury and dysfunction, which is an initial step of atherosclerosis. Fibrinopeptide A (FPA) is a biomarker of the activation of the coagulation system, and a high concentration of FPA in the blood occurs in patients with ischemic cardiocerebrovascular diseases. The present research observed that FPA stimulated the generation of C-reactive protein (CRP), IL-1β, and IL-6 in human umbilical vascular endothelial cells (HUVECs); and anti-IL-1 β and anti-IL-6 neutralizing antibodies did not alter FPA-induced CRP expression in HUVECs. The subchronic administration of FPA into rats increased the plasma FPA and CRP levels. Further studies showed that FPA stimulated superoxide anion generation, activated ERK1/2 and p38, promoted nuclear factor κB (NF-κB) nuclear translocation, and raised the NF-κB level in the nuclei of HUVECs. Antioxidant N-acetylcysteine (NAC), complex II inhibitor thenoyltrifluoroacetone (TTFA), and NADPH oxidase inhibitor diphenyleneiodonium (DPI) inhibited FPA-stimulated generation of superoxide anion, and NAC reduced FPA-induced expressions of the phosphorylated ERK1/2 and p38. NAC, TTFA, DPI, inhibitors of ERK1/2, p38, and NF-κB all downregulated FPA-induced CRP expression. These results indicate that FPA induces CRP expression in HUVECs via the ROS-ERK1/2/p38-NF-κB signal pathway. Moreover, this is the first report that FPA produces a proinflammatory effect on the vascular endothelial cells.     

Characterizing the Mechanical Variations of Human Femoropopliteal Artery During Aging Process

Vascular diseases during aging process are closely correlated to the age-related changes of mechanical stimuli for resident cells. Characterizing the variations of mechanical environments in vessel walls with advancing age is crucial for a better understanding of vascular remodeling and pathological changes. In this study, the mechanical stress, strain, and wall stiffness of the femoropopliteal arteries (FPAs) were compared among four different age groups from adolescent to young, middle-aged, and aged subjects. The material parameters and geometries adopted in the FPA models were obtained from published experimental results. It is found that high mechanical stress appears at different layers in young and old FPA walls respectively. The characteristics of the middle-aged FPA wall suggests that it is the most capable of resisting high blood pressures and maintaining a mechanical homeostasis during the entire life span. It is demonstrated that the variations of stress and strain rather than that of wall stiffness can be used as an indicator to illustrate the profile of FPA aging. Our results could serve as an age-specific mechanical reference for vascular mechanobiological studies, and allow further exploration of cellular dysfunctions in vessel walls during aging process.     

The Fluid Processing Apparatus: from flight hardware to electron micrographs

Sweet clover seeds were grown in the Fluid Processing Apparatus (FPA) flown on STS-54 (January 1993) and STS-60 (February 1994). After germination and seedling growth for 40 hours, seedlings were fixed in space. Electron microscopy was used to examine the seedling ultrastructure to verify that fixation occurred and preserved the samples. Micrographs revealed well-preserved cell structure and the presence of calcium precipitates indicating complete fixation. FPA is a useful piece of hardware for preservation/fixation during spaceflight.     

Evidence that p-fluorophenylalanine has a direct effect on tubulin in Aspergillus nidulans.

Three temperature-sensitive alleles of benA (benA11, 17 and 21) confer resistance to growth inhibition by p-fluorophenylalanine (FPA). FPA resistance cosegregates with the benA gene. Two back-mutations in benA which cause loss of temperature sensitivity cause loss of FPA resistance, and two indirect suppressors of benA temperature sensitivity also cause FPA resistance to be lost. These results indicate that FPA resistance is an intrinsic property of the benA mutations. The intracellular phenylalanine concentrations of these strains are normal as is their ability to take up phenylalanine from the medium. We conclude that FPA must inhibit growth and cause non-disjunction by a direct effect on the polymerization of tubulin.     

Induction of capsular polysaccharide synthesis by rho-fluorophenylalanine in Escherichia coli wild type and strains with altered phenylalanyl soluble ribonucleic acid synthetase

Escherichia coli K-12 strain AB259 can be induced to form capsular polysaccharide (mucoid clones) by dl-p-fluorophenylalanine (FPA; 5 x 10(-6)m on agar plates at 37 C or 8 x 10(-5)m in liquid medium at 30 C). The change was shown to be phenotypic. An increase in enzymes probably involved in capsular polysaccharide synthesis [phosphomannose isomerase (3.3-fold), uridine diphosphate-d-galactose-4-epimerase (2.5-fold), and guanine diphosphate-l-fucose synthetase] was demonstrated as a result of growth in FPA. These increases appear sufficient to account for the increased synthesis of capsular polysaccharide due to growth in FPA. FPA-resistant derivatives of strain AB259 were obtained by selecting mutants on FPA-containing agar or by transducing in an altered phenylalanyl soluble ribonucleic acid synthetase that activates FPA poorly. Mucoid clones were formed by these strains only in the presence of 30 to 1,000 times as much FPA. Among these strains, there was a close correlation between incorporation of FPA-C(14) and induction of capsular polysaccharide synthesis. The results are thus consistent with the following model: FPA is incorporated into the protein product of the R(1) gene (repressor) and alters it sufficiently to allow derepression of several enzymes.     

Allosteric effects potentiating the release of the second fibrinopeptide A from fibrinogen by thrombin

Fibrin formation depends on the release of the two N-terminal fibrinopeptides A (FPA) from fibrinogen, and its formation is accompanied by an intermediate, alpha-profibrin, which lacks only one of the FPA. In this study, we confirm that the maximal levels of alpha-profibrin found over the course of thrombin reactions with human fibrinogen are only half of what would be expected if the first and second FPA were being released independently with equal rate constants. The rapidity of release of the fibrinopeptides by thrombin had been shown to depend on an allosteric transformation that is induced when Na(+) binds to a site defined by the 215-227 residues of thrombin, a transformation that results in the exposure of its fibrinogen-binding exosites transforming the thrombin from a slow to a fast acting form toward fibrinogen. When choline was substituted for sodium to transform thrombin to its slow form, the maximal levels of alpha-profibrin rose to those expected for independent release of the two FPA. Thus, it is only the fast thrombin that releases the second FPA fast, and that fast release only occurs when both FPA are present because of a partial coupling of its release with that of the first FPA. The release of the FPA from purified alpha-profibrin with the first FPA already missing is no faster than the release of any FPA. Surprisingly, we also found that slow thrombin became increasingly transformed to a fast form in the absence of sodium when the fibrinogen was elevated to high concentrations. This potentiation by concentrated fibrinogen also occurs with the recombinant mutant thrombin (Y225P), which is otherwise slow in both the presence and absence of Na(+). The potentiation of thrombin by fibrinogen must be short-lived so that the thrombin reverts to its slow acting form in the interim among encounters with other fibrinogen molecules in dilute fibrinogen solutions lacking Na(+), whereas at high fibrinogen concentrations the thrombin encounters other molecules before it reverts back to the slow form.     

Inhibition of Leaf Processes by p-Fluorophenylalanine During Induction of Flowering in the Cocklebur

The amino acid antimetabolite, DL-p-fluorophenylalanine (FPA), inhibited induction of flowering in the short-day cocklebur plant, Xanthium pensylvanicum Wall., primarily by interfering with processes occurring during the inductive dark period. At the concentrations used the inhibitor had little effect on subsequent vegetative development of the plant.The inhibition was largely reversed (internally) by L-phenylalanine, but not by D-phenylalanine nor by DL-tyrosine. The FPA strongly inhibited the absorption of labeled phenylalanine, leucine, and glycine, and inhibited the conversion of phenylalanine into protein in experiments where incorporation was separated in time from effects upon absorption. The FPA, too, was incorporated into protein, at nearly half the rate of phenylalanine. Neither D- nor L-phenylalanine significantly interfered with absorption of FPA, showing the FPA did not affect amino acid absorption by simple competition for a common carrier site. It was concluded that FPA may affect flower induction because of its interference with normal enzyme synthesis, although effects on other processes might also be involved.     

Validity and reliability of a shoe-embedded sensor module for measuring foot progression angle during over-ground walking

Wearable systems are becoming increasingly popular for gait assessment outside of laboratory settings. A single shoe-embedded sensor module can measure the foot progression angle (FPA) during walking. The FPA has important clinical utility, particularly in populations with knee osteoarthritis, as it is a target for biomechanical treatments. However, the validity and the day-to-day reliability of FPA measurement using wearable systems during over-ground walking has yet to be established. Two gait analysis sessions on 20 healthy adults were conducted. During both sessions, participants performed natural over-ground walking in a motion capture laboratory and on a 100 m linear section of outdoor athletics track. FPA was measured in the laboratory via marker trajectory data, while the sensor module measured FPA during the outdoor track walking. Validity was examined by comparing the laboratory- and sensor-measured average FPA. Day-to-day reliability was examined by comparing the sensor-measured FPA between the first and second gait analysis sessions. Average absolute error between motion capture and sensor measured FPA were 1.7° and 2.1° at session 1 and 2, respectively. A Bland and Altman plot indicated no systematic bias, with 95% limit of agreement widths of 4.2° – 5.1°. Intraclass correlation coefficient (ICC_2k) analysis resulted in good to excellent validity (ICC = 0.89 – 0.91) and reliability (ICC = 0.95). Overall, the shoe-embedded sensor module is a valid and reliable method of measuring FPA during over-ground walking without the need for laboratory equipment.     

High-Accuracy Brain-Machine Interfaces Using Feedback Information

Sensory feedback is very important for movement control. However, feedback information has not been directly used to update movement prediction model in the previous BMI studies, although the closed-loop BMI system provides the visual feedback to users. Here, we propose a BMI framework combining image processing as the feedback information with a novel prediction method. The feedback-prediction algorithm (FPA) generates feedback information from the positions of objects and modifies movement prediction according to the information. The FPA predicts a target among objects based on the movement direction predicted from the neural activity. After the target selection, the FPA modifies the predicted direction toward the target and modulates the magnitude of the predicted vector to easily reach the target. The FPA repeats the modification in every prediction time points. To evaluate the improvements of prediction accuracy provided by the feedback, we compared the prediction performances with feedback (FPA) and without feedback. We demonstrated that accuracy of movement prediction can be considerably improved by the FPA combining feedback information. The accuracy of the movement prediction was significantly improved for all subjects (P<0.001) and 32.1% of the mean error was reduced. The BMI performance will be improved by combining feedback information and it will promote the development of a practical BMI system.     

Microplate-based filter paper assay to measure total cellulase activity

The standard filter paper assay (FPA) published by the International Union of Pure and Applied Chemistry (IUPAC) is widely used to determine total cellulase activity. However, the IUPAC method is not suitable for the parallel analyses of large sample numbers. We describe here a microplate-based method for assaying large sample numbers. To achieve this, we reduced the enzymatic reaction volume to 60 microl from the 1.5 ml used in the IUPAC method. The modified 60-microl format FPA can be carried out in 96-well assay plates. Statistical analyses showed that the cellulase activities of commercial cellulases from Trichoderma reesei and Aspergillus species determined with our 60-microl format FPA were not significantly different from the activities measured with the standard FPA. Our results also indicate that the 60-microl format FPA is quantitative and highly reproducible. Moreover, the addition of excess beta-glucosidase increased the sensitivity of the assay by up to 60%.     

Formation of soluble fibrin oligomers in purified systems and in plasma.

The kinetic parameters for release of fibrinopeptide A (FPA) from human fibrinogen by thrombin are: Km = 2.3 X 10(-6)M and Vmax. = 1.1 X 10(-10)mol of FPA/s per unit of thrombin; for fibrin formation, Km is similar to that for FPA release, but, the conditions of the present study, Vmax. was approximately half of that for FPA release. The formation of fibrin polymer before the sol-gel transition was studied by gel-permeation chromatography combined with effluent analysis for fibrinogen antigen and residual FPA. Polymer formation in purified fibrinogen incubated with thrombin proceeded as a bimolecular association of exposed sites in a manner predicted by probability calculations and assuming random FPA cleavage. Each oligomer consisted of n molecules of fibrin monomer and two fibrinogen molecules, each of the latter lacking one FPA molecule, i.e. each oligomer, regardless of molecular size, retains two FPA molecules. The addition of 5 mM-CaCl2 to the reaction mixture changed the rate of polymer formation, so that dimer was no longer the prevalent oligomer; in the presence of Ca2+, the trimer was the oligomer in highest concentration. The polymers formed in the presence of calcium were similar in composition to those without, i.e. 2 mol of FPA/mol of oligomer. EDTA-treated plasma samples incubated for short periods of time, 30s or less, with thrombin ranging in concentration up to 1 N.I.H. unit/ml did not form clots during the 10-15 min period of observation until they were applied to the column, though a large proportion of the available FPA was cleaved (maximum 45%). The soluble polymers in plasma were mostly of the high-Mr variety (tetramer and greater); these high-Mr polymers contained less than 2 mol of FPA/mol of polymer, whereas dimer and trimer in plasma were similar to those in the purified systems, i.e. 2 mol of FPA/mol.     

FPA, a gene involved in floral induction in Arabidopsis, encodes a protein containing RNA-recognition motifs

FPA is a gene that regulates flowering time in Arabidopsis via a pathway that is independent of daylength (the autonomous pathway). Mutations in FPA result in extremely delayed flowering. FPA was identified by means of positional cloning. The predicted FPA protein contains three RNA recognition motifs in the N-terminal region. FPA is expressed most strongly in developing tissues, similar to the expression of FCA and LUMINIDEPENDENS, two components of the autonomous pathway previously identified. Overexpression of FPA in Arabidopsis causes early flowering in noninductive short days and creates plants that exhibit a more day-neutral flowering behavior.     

Accessory enzymes influence cellulase hydrolysis of the model substrate and the realistic lignocellulosic biomass

The potential of cellulase enzymes in the developing and ongoing “biorefinery” industry has provided a great motivation to develop an efficient cellulase mixture. Recent work has shown how important the role that the so-called accessory enzymes can play in an effective enzymatic hydrolysis. In this study, three newest Novozymes Cellic CTec cellulase preparations (CTec 1/2/3) were compared to hydrolyze steam pretreated lignocellulosic substrates and model substances at an identical FPA loading. These cellulase preparations were found to display significantly different hydrolytic performances irrelevant with the FPA. And this difference was even observed on the filter paper itself when the FPA based assay was revisited. The analysis of specific enzyme activity in cellulase preparations demonstrated that different accessory enzymes were mainly responsible for the discrepancy of enzymatic hydrolysis between diversified substrates and various cellulases. Such the active role of accessory enzymes present in cellulase preparations was finally verified by supplementation with β-glucosidase, xylanase and lytic polysaccharide monooxygenases AA9. This paper provides new insights into the role of accessory enzymes, which can further provide a useful reference for the rational customization of cellulase cocktails in order to realize an efficient conversion of natural lignocellulosic substrates.     

Cellular compartmentation of aromatic amino acids in Neurospora crassa. I. Occupation of a protein synthesis pool by phenylalanine in Tyr-1 mutants

The p-fluorophenylalanine (FPA) resistance of acc ^phe, which has previously been shown (Brooks et al., 1972) to be a try-1 mutant, has been further investigated. When incubated in the absence of tyrosine, acc ^phe and also tyr-1 auxotrophs show a gradual increase in free phenylalanine in the cell but a sharp decrease in FPA incorporation into protein. The decrease in FPA incorporation is apparently due to the excess phenylalanine in the mutants, since the normal endogenous pool component in wild type and also in the mutants incubated on tyrosine does not appear to compete with FPA for incorporation. The rate of FPA incorporation into protein in acc ^phe remains at 10–15% of the wild-type rate even when the ratio of free FPA to excess phenylalanine in the cell is high as 8:1. If wild type is supplied with exogenous phenylalanine and FPA simultaneously, phenylalanine is preferentially incorporated into protein but, in contrast to the mutant, the rate of FPA incorporation increases as the ratio of free FPA to phenylalanine increases. On the basis of differences in competition with FPA and in susceptibilities to mild extraction procedures, it is proposed that phenylalanine can be located in at least three compartments in Neurospora: a small constant-size endogenous pool always seen in wild type; an expandable exogenous pool; and a protein synthesis pool which is preferentially populated by endogenous phenylalanine but can be entered by exogenous molecules when biosynthesis is regulated. In acc ^phe, where phenylalanine biosynthesis is not regulated, the excess phenylalanine is located primarily in the protein synthesis pool where it only has to compete with a small FPA component and is thereby preferentially incorporated into protein in this mutant.This work was supported, in part, by an Atomic Energy Commission grant to the Institute of Molecular Biophysics, The Florida State University, and by the Genetics Training Grant, funded by the National Institutes of Health. It contains, in part, data from the doctoral thesis of the senior author, who was supported by a Florida State University Nuclear Fellowship and by a Public Health Service Fellowship.     

Evaluation of the fluorescence polarization assay and comparison to other serological assays for detection of brucellosis in cervids

The complement fixation test (CFT), competitive enzyme immunoassay (CELISA), indirect enzyme immunoassay (IELISA) and fluorescence polarization assay (FPA) were evaluated for the detection of antibodies to Brucella abortus and Brucella suis biotype 4 in caribou (Rangifer tarandus caribou), elk (Cervus elapus), red deer (Cervus elapus), and reindeer (Rangifer tarandus tarandus). When combining the data the FPA and the CELISA were determined to be the most suitable tests for serodiagnosis of Cervidae. The overall actual sensitivity of the CFT and the IELISA was 100%. The overall actual sensitivity for the CELISA and FPA was 99%. The overall relative specificity of the CFT (including treatment of anti-complementary data as positive or negative for analysis), the CELISA, the IELISA and the FPA were 65%, 93%, 99%, 99%, and 99%, respectively. The specificities of the buffered plate agglutination test (BPAT), the CFT, the CELISA, the FPA and the IELISA for 55 elk vaccinated with B. abortus strain 19 and tested 4 mo post vaccination were 14%, 31%, 51%, 84%, and 2%, respectively. The FPA is the diagnostic test of choice because it has sensitivity and specificity values comparable to the CELISA; it has the capability to distinguish vaccinal antibody and antibody resulting from exposure to cross-reacting organisms such as Yersinia enterocolitica 0:9 from antibody to Brucella spp. in most cases; it is technically simple to do; it is adaptable to field use and it is relatively inexpensive.     

Standardization of the filter paper activity assay for solid substrate fermentation

The conditions of the filter paper activity (FPA) assay were standardized for solid substrate fermentation (SSF). The FPA is a relative measure of the overall cellulose hydrolysing capacity of microbial cellulase preparations, thus reliable and comparable data may be obtained only under standardized conditions. The standardization developed for submerged fermentation (SF) cannot be translated directly to SSF. In SSF, the FPA is strongly dependent on the extraction volume and on the dilution of the enzyme in the assay. The optimal extraction volume was substrate dependent in SSF of corn fiber, spent brewing grains and wheat straw for cellulase production by Trichoderma reesei Rut C30. Other cellulolytic enzyme assays (endoglucanase, beta-glucosidase and xylanase) were much less sensitive to the extraction volume.     

Measurement of filter paper activities of cellulase with microplate-based assay

It is always a challenge to determine the total cellulase activity efficiently without reducing accuracy. The most common total cellulase activity assay is the filter paper assay (FPA) established by the International Union of Pure and Applied Chemistry (IUPAC). A new procedure to measure the FPA with microplate-based assay was studied in this work, which followed the main idea of IUPAC to dilute cellulase preparation to get fixed glucose release. FPAs of six cellulase preparations were determined with the microplate-based assay. It is shown that FPAs of cellulase Youtell, RCconc, R-10, Lerkam, Yishui and Sinopharm were 67.9, 46.0, 46.1, 27.4, 7.6 and 8.0 IU/ml respectively. There was no significant difference at the 95% confidence level between the FPA determined with IUPAC and the microplate-based assay. It could be concluded that the FPA could be determined by the microplate-based assay with the same accuracy and much more efficiency compared with that by IUPAC.     

Thrombin-induced fibrinopeptide release from a fibrinogen variant (fibrinogen Sydney I) with an Aalpha Arg-16----His substitution

Fibrinogen, purified from a recently identified case of dysfibrinogenaemia, fibrinogen Sydney I, was shown by thrombin digestion, high-performance liquid chromatography (HPLC) and amino acid analysis to be a heterozygous case of an A alpha Arg-16----His substitution. Kinetic studies have been carried out on the thrombin-induced release of fibrinopeptide A (FPA), fibrinopeptide B (FPB) and the variant peptide [His16]FPA. When thrombin was added to fibrinogen Sydney I at a concentration of 0.2 U/ml release of FPA was rapid and there was a 79-fold reduced rate of release of [His16]FPA, but the rate of release of FPB was not appreciably reduced. In contrast, at lower thrombin concentrations the rate of FPB release was reduced in proportion to the rate of total FPA release, supporting the view that release of fibrinopeptides is a sequential process. The second-order kinetic constant kcat/Km for hydrolysis of the abnormal A alpha chain by thrombin was calculated from Lineweaver-Burk plots to be 16-30-fold less than that for the normal A alpha chain. Molecular modelling studies, using a refined model of the trypsin-pancreatic-trypsin-inhibitor complex have been used to suggest how the histidine at the P1 site can be accommodated within the enzyme hydrophobic active-site pocket.     

The activity of postural asymmetry factors in symmetrical sections of the rat spinal cord

The distribution of the low-molecular weight and high-molecular weight postural asymmetry factors (FPA) activity in the left and right parts of the lumbal region of the rat spinal cord was studied. Low-molecular weight FPA induces flexion of the hind limb ipsilateral to the half of the spinal cord from which FPA was isolated, while high-molecular weight FPA induces contralateral flexion. The activities of the low- and high-molecular weight FPAs in each half of the spinal cord are comparable in normal rat. After the suction lesion of the motor areas in the left hemisphere the increase of the low-molecular weight FPA activity in the right half of the lumbal region of the spinal cord was observed.