Effect of dynamic stability on a step task in ACL deficient individuals.

Stair ascent and descent requires large knee motions and muscle forces that can be challenging for people with anterior cruciate ligament (ACL) deficiency. Movement and muscle activity patterns were compared in two groups of ACL deficient subjects and a group of uninjured subjects. The ACL deficient subjects were prospectively classified according to functional ability. "Copers" were defined as individuals with complete ACL rupture and no symptoms of knee instability and participated in high-level sports without difficulty. "Non-copers" were defined as ACL deficient individuals who had instability with low-level daily activities and were not able to participate in sports. Sagittal plane kinematic and kinetic data from the hip, knee and ankle and electromyographic data from the vastus lateralis, lateral hamstring, medial gastrocnemius, and soleus were collected as subjects stepped up and over a 26 cm high step. Both coper and non-coper subjects had altered movement patterns as they controlled the rapid movement from step ascent to descent with their involved limbs. Only non-copers used significantly different movement patterns on their involved limb compared to controls after they had descended from the step and their involved side accepted the weight of the body. Classifying subjects by functional ability resulted in more pronounced differences in movement patterns between non-copers and copers. Copers moved more like uninjured subjects.     

Quadriceps femoris muscle morphology and function after ACL injury: a differential response in copers versus non-copers

The morphology (volume and peak cross-sectional area) and voluntary muscle control of 27 athletic people were evaluated with magnetic resonance imaging (MRI) and an established method of testing neuromuscular control in order to explain why some people are able to cope with anterior cruciate ligament (ACL) injury (copers), whereas most cannot (non-copers). Axial spin-echo T1 weighted MRI images were acquired from the level of the ankle mortise to the iliac crest. The subjects' quadriceps, hamstrings, and gastrocnemius muscles were digitally reconstructed from the MRI images. The volume and peak cross-sectional area (CSA) of each muscle were then calculated. Voluntary muscle control was evaluated using an established target-matching protocol that requires subjects to produce and modulate force with control over a range of directions. Electromyographic signals were collected from seven muscles as the subjects performed the experiment. Circular statistics methods were used to calculate a specificity index that describes how focused the activity pattern of each muscle was with respect to its principal direction of action. The results of the non-copers, copers, and uninjured subjects were then compared. The non-copers displayed significantly greater quadriceps atrophy than the copers. The most profound differences were observed in the vastus lateralis muscle. The non-copers also displayed diminished vastus lateralis and lateral gastrocnemius muscle control. Little differences were observed in the results of the copers and uninjured subjects. In general, the copers' results fell between those of the non-copers and uninjured subjects. The results of this study suggest that quadriceps muscle function is a critical factor in the differential response to ACL injury.     

Co-contraction during static and dynamic knee extensions in ACL deficient subjects.

Co-contraction of the muscles is proposed in the literature as one of the strategies that anterior cruciate ligament deficient (ACLD) subjects can use to compensate the loss of ACL function. This study examined the response of ACLD and control subjects to different shear forces in isometric and slow-dynamic knee extensions. Twelve chronic ACLD and 10 control subjects performed submaximal positioning and slow-dynamic knee extensions (between 45 degrees and 5 degrees of knee flexion) with two external flexion moments both applied at two distances on the lower leg. The shear force was controlled by changing the moment arm without changing the moment. Electromyographic data were collected from knee flexor and extensor muscles. In the analysis of variance, no significant effect of subject group was found in positioning or slow-dynamic tasks across all muscles. The effect of knee angle was significantly different between the subject groups for biceps femoris in positioning and for rectus femoris in slow-dynamic tasks, but these effects were very small and will not have a great impact on the resulting shear forces. There was no interaction between moment arm and subject group. Therefore, the hypothesis that ACLD subjects increase co-contraction in situations with an increased shear load in positioning and slow-dynamic knee extensions could not be confirmed.     

Gait adaptation in ACL deficient patients before and after anterior cruciate ligament reconstruction surgery.

The objective of this study is to determine how kinematical parameters and electromyography data of selected muscles may change as a result of anterior cruciate ligament (ACL) deficiency and following ACL reconstruction. The study was conducted on 25 anterior cruciate ligament deficient subjects prior to and 6 weeks, 4 months, 8 months and 12 months following ACL reconstructive surgery using the bone-patellar tendon-bone technique. Gait analysis was performed by applying the zebris three-dimensional ultrasound-based system with surface electromyograph (zebris). Kinematic data were recorded for the lower limb. The muscles surveyed include vastus lateralis and medialis, biceps femoris and adductor longus. The results obtained from the injured subjects were compared with those of 51 individuals without any ACL damage whatsoever. Acute ACL deficient patients exhibited a quadriceps avoidance pattern prior to and 6 weeks following surgery. No quadriceps avoidance phenomenon develops in chronic ACL deficient patients. In operated individuals, tempo-spatial parameters and the knee angle regained a normal pattern for the ACL-deficient limb during gait as early as 4 months following surgery. However, the relative ACL movement parameter, which describes the tibial translation into the direction of ACL, and the EMG traces show no significant statistical difference compared with the same values of the healthy control group just 8 months following surgery. The analysis of spatial-temporal parameters and EMG traces show that the development of a quadriceps avoidance pattern is less common than previously reported. These data suggest that anterior cruciate ligament deficiency and reconstruction produce considerable changes in the lower extremity gait pattern. The results suggest that gait parameters tend to shift towards a normal value pattern; and the re-establishment of pre-injury gait patterns-including the normal biphase of muscles-takes at least 8 months to occur.     

Reduced knee joint moment in ACL deficient patients at a cost of dynamic stability during landing

The current study aimed to examine the effect of anterior cruciate ligament deficiency (ACLd) on joint kinetics and dynamic stability control after a single leg hop test (SLHT). Twelve unilateral ACLd patients and a control subject group (n=13) performed a SLHT over a given distance with both legs. The calculation of joint kinetics was done by means of a soft-tissue artifact optimized rigid full-body model. Margin of stability (MoS) was quantified by the difference between the base of support and the extrapolated center of mass. During landing, the ACLd leg showed lower external knee flexion moments but demonstrated higher moments at the ankle and hip compared to controls (p<0.05). The main reason for the joint moment redistribution in the ACLd leg was a more anterior position of the ground reaction force (GRF) vector, which affected the moment arms of the GRF acting about the joints (p<0.05). For the ACLd leg, trunk angle was more flexed over the entire landing phase compared to controls (p<0.05) and we found a significant correlation between moment arms at the knee joint and trunk angle (r2 = 0.48;p<0.01). The consequence of this altered landing strategy in ACLd legs was a more anterior position of the center of mass reducing the MoS (p<0.05). The results illustrate the interaction between trunk angle, joint kinetics and dynamic stability during landing maneuvers and provide evidence of a feedforward adaptive adjustment in ACLd patients (i.e. more flexed trunk angle) aimed at reducing knee joint moments at the cost of dynamic stability control.     

Frequency content asymmetry of the isokinetic curve between ACL deficient and healthy knee

The torque-time curve patterns of concentric isokinetic knee extension in anterior cruciate ligament (ACL) deficient patients usually present mid-range irregularities associated with the level of anterior tibial translation. The purpose of this study was to compare the smoothness in isokinetic torque production between the ACL deficient and the healthy knee. Thirty ACL deficient soccer players performed bilaterally five trials of maximum concentric knee extension-flexion at 60 degrees /s on a Biodex dynamometer. The three middle trials (a total of six curves) were retained and submitted to further data processing. Maximum frequency values contained within the 90%, 95% and 99% level of the signal power were calculated for each extension and flexion curve. The frequency content of the ACL deficient side proved to be statistically higher compared to the intact side at all levels of the power spectrum. The percentage differences in the frequency content were 18.8%, 10.6% and 40.0% for knee extension, and 49.5%, 24.5% and 16.3% for knee flexion, for the respective power levels. This indicated higher oscillations and, therefore, more unstable mechanical output of the injured knee. An overall biological interpretation of the present results is based on the notion that disturbed motion is generally connected to poor level of joint functionality.     

Inhibition of AmpC beta-lactamase through a destabilizing interaction in the active site.

Beta-lactamases hydrolyze beta-lactam antibiotics, including penicillins and cephalosporins; these enzymes are the most widespread resistance mechanism to these drugs and pose a growing threat to public health. beta-Lactams that contain a bulky 6(7)alpha substituent, such as imipenem and moxalactam, actually inhibit serine beta-lactamases and are widely used for this reason. Although mutant serine beta-lactamases have arisen that hydrolyze beta-lactamase resistant beta-lactams (e.g., ceftazidime) or avoid mechanism-based inhibitors (e.g., clavulanate), mutant serine beta-lactamases have not yet arisen in the clinic with imipenemase or moxalactamase activity. Structural and thermodynamic studies suggest that the 6(7)alpha substituents of these inhibitors form destabilizing contacts within the covalent adduct with the conserved Asn152 in class C beta-lactamases (Asn132 in class A beta-lactamases). This unfavorable interaction may be crucial to inhibition. To test this destabilization hypothesis, we replaced Asn152 with Ala in the class C beta-lactamase AmpC from Escherichia coli and examined the mutant enzyme's thermodynamic stability in complex with imipenem and moxalactam. Consistent with the hypothesis, the Asn152 --> Ala substitution relieved 0.44 and 1.10 kcal/mol of strain introduced by imipenem and moxalactam, respectively, relative to the wild-type complexes. However, the kinetic efficiency of AmpC N152A was reduced by 6300-fold relative to that of the wild-type enzyme. To further investigate the inhibitor's interaction with the mutant enzyme, the X-ray crystal structure of moxalactam in complex with N152A was determined to a resolution of 1.83 A. Moxalactam in the mutant complex is significantly displaced from its orientation in the wild-type complex; however, moxalactam does not adopt an orientation that would restore competence for hydrolysis. Although Asn152 forces beta-lactams with 6(7)alpha substituents out of a catalytically competent configuration, making them inhibitors, the residue is essential for orienting beta-lactam substrates and cannot simply be replaced with a much smaller residue to restore catalytic activity. Designing beta-lactam inhibitors that interact unfavorably with this conserved residue when in the covalent adduct merits further investigation.     

UGUS, a reporter for use with destabilizing N-termini.

Due to constraints in vector construction, reporter polypeptides often carry N-terminal sequences of extraneous origin. Since protein half-life can be influenced by small determinants in the N-terminus, such foreign sequences can destabilize proteins and compromise results of reporter-based studies. We provide a real-life example of this problem (destabilizing sequences derived from a ribosomal protein) and show that it can be solved with the ubiquitin fusion technique, in which ubiquitin sequences are placed upstream of the reporter, in our case beta-glucuronidase. Post-translational processing by characterized pathways removes the ubiquitin together with destabilizing sequences, generating a stable reporter whose N-terminus is constant.     

Tolerance of T4 lysozyme to proline substitutions within the long interdomain alpha-helix illustrates the adaptability of proteins to potentially destabilizing lesions.

To investigate the ability of a protein to accommodate potentially destabilizing amino acid substitutions, and also to investigate the steric requirements for catalysis, proline was substituted at different sites within the long alpha-helix that connects the amino-terminal and carboxyl-terminal domains of T4 lysozyme. Of the four substitutions attempted, three yielded folded, functional proteins. The catalytic activities of these three mutant proteins (Q69P, D72P, and A74P) were 60-90% that of wild-type. Their melting temperatures were 7-12 degrees C less than that of wild-type at pH 6.5. Mutant D72P formed crystals isomorphous with wild-type allowing the structure to be determined at high resolution. In the crystal structure of wild-type lysozyme the interdomain alpha-helix has an overall bend angle of 8.5 degrees. In the mutant structure the introduction of the proline causes this bend angle to increase to 14 degrees and also causes a corresponding rotation of 5.5 degrees of carboxyl-terminal domain relative to the amino-terminal one. Except for the immediate location of the proline substitution there is very little change in the geometry of the interdomain alpha-helix. The results support the view that protein structures are adaptable and can compensate for potentially destabilizing amino acid substitutions. The results also suggest that the precise shape of the active site cleft of T4 lysozyme is not critical for catalysis.     

Elucidation of enzyme control mechanisms using macroscopic measurements in a mixed substrate fermentation system

The objective of this work was to relate macroscopically measurable on-line fermentation parameters such as dissolved oxygen, off-gas oxygen and carbon dioxide, and cell mass, to the controlled production of key intracellular enzymes under carbon limited conditions. Both batch and perturbed batch aerobic fermentations were performed using two different strains of Escherichia coli, with glucose and lactose as the sole carbon sources. The two strains differed from each other only in the lac operon region of their genome. The parent strain, E. coli 3000, was inducible for the enzyme beta-galactosidase. The other strain, E. coli 3300, was a constitutive mutant in the production of beta-galactosidase. In all experiments, off-line assays of sugars and beta-galactosidase activity were performed. It was observed that there is a clear relationship between the macroscopic on-line measurements, dissolved oxygen tension, carbon dioxide evolution rate and oxygen uptake rate, and the microscopic control phenomena of catabolite repression, catabolite inhibition, and inducer repression.     

Structural and genetic analysis of a mutant of Rhodobacter sphaeroides WS8 deficient in hook length control.

Motility in the photosynthetic bacterium Rhodobacter sphaeroides is achieved by the unidirectional rotation of a single subpolar flagellum. In this study, transposon mutagenesis was used to obtain nonmotile flagellar mutants from this bacterium. We report here the isolation and characterization of a mutant that shows a polyhook phenotype. Morphological characterization of the mutant was done by electron microscopy. Polyhooks were obtained by shearing and were used to purify the hook protein monomer (FlgE). The apparent molecular mass of the hook protein was 50 kDa. N-terminal amino acid sequencing and comparisons with the hook proteins of other flagellated bacteria indicated that the Rhodobacter hook protein has consensus sequences common to axial flagellar components. A 25-kb fragment from an R. sphaeroides WS8 cosmid library restored wild-type flagellation and motility to the mutant. Using DNA adjacent to the inserted transposon as a probe, we identified a 4.6-kb SalI restriction fragment that contained the gene responsible for the polyhook phenotype. Nucleotide sequence analysis of this region revealed an open reading frame with a deduced amino acid sequence that was 23.4% identical to that of FliK of Salmonella typhimurium, the polypeptide responsible for hook length control in that enteric bacterium. The relevance of a gene homologous to fliK in the uniflagellated bacterium R. sphaeroides is discussed.     

Conditional control of chimeric antigen receptor T-cell activity through a destabilizing domain switch and its chemical ligand

Background aimsDespite the impressive efficacy of chimeric antigen receptor (CAR) T-cell therapy, adverse effects, including cytokine release syndrome and neurotoxicity, impede its therapeutic application, thus making the modulation of CAR T-cell activity a priority. The destabilizing domain mutated from Escherichia coli dihydrofolate reductase (DHFR) is inherently unstable and degraded by proteasomes unless it is stabilized by its chemical ligand trimethoprim (TMP), a Food and Drug Administration-approved drug. Here the authors reveal a strategy to modulate CAR T-cell activity at the protein level by employing DHFR and TMP as a chemical switch system.MethodsFirst, the system was demonstrated to work in human primary T cells. To introduce the system to CAR T cells, DHFR was genetically fused to the carboxyl terminal of a third-generation CAR molecule targeting CD19 (CD19-CAR), constructing the CD19-CAR-DHFR fusion.ResultsThe CD19-CAR-DHFR molecule level was shown to be modulated by TMP. Importantly, the incorporation of DHFR had no impact on the recognition specificity and normal function of the CAR molecule. Little adverse effect on cell proliferation and apoptosis was detected. It was proved that TMP could regulate cytokine secretion and the in vitro cytotoxicity of CD19-CAR-DHFR T cells. Furthermore, the in vivo anti-tumor efficacy was demonstrated to be controllable through the manipulation of TMP administration. The approach to control CD19-CAR also succeeded in 19-BBZ(71), another CD19-targeting CAR with a different structure.ConclusionsThe proposed approach based on DHFR and TMP provides a facile strategy to bring CAR T-cell therapy under conditional user control, and the strategy may have the potential to be transplantable.     

Elucidation of a novel polypeptide cross-link involving 3-hydroxykynurenine.

3-Hydroxykynurenine, a metabolite of tryptophan, is a powerful antioxidant and neurotoxin. The neurotoxicity results from the oxidation of 3-hydroxykynurenine, and hydroxyl radicals, formed via H(2)O(2), may also be implicated [Okuda, S., Nishiyama, N., Saito, H. , and Katsuki, H. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 12553-12558]. Oxidation of o-aminophenols, such as 3-hydroxykynurenine, also results in the formation of highly reactive quinonimines. Thus, one possible consequence of 3-hydroxykynurenine oxidation may be covalent modification of cellular macromolecules. Such a process could contribute to the neurotoxicity and may potentially be important in other tissues, such as the human lens, where 3-hydroxykynurenine functions as a UV filter. In this work, we demonstrate that 3-hydroxykynurenine can bind to protein amino groups and, further, that under oxidative conditions, 3-hydroxykynurenine can function to cross-link polypeptide chains. The structure of the cross-linked moiety, using the peptide glycyllysine, has been elucidated. The cross-link, which is both colored and fluorescent, involves the peptide alpha-amino groups. Proteins modified by 3-hydroxykynurenine become colored and fluorescent as well as cross-linked. LC-MS studies indicate that the cross-link is also present in gamma-crystallin, following incubation of this lens protein in the presence of 3-hydroxykynurenine. Similar posttranslational modifications of lens proteins accompany cataract formation, and knowledge of the precise mode of reaction of 3-hydroxykynurenine with proteins will assist in determining if 3-hydroxykynurenine is involved in degenerative conditions in which oxidation of such aminophenols is implicated.     

Synthesis and biological evaluation of a library containing potentially 1600 amides / esters. A strategy for rapid compound generation and screening.

A library of potentially 1600amide/ester dimers was prepared by reacting 40 acid chlorides with 40 nucleophiles. The whole library was presented for biological screening in 80 sample mixtures, with each of the possible products appearing in a unique pair of samples. The potential of this strategy for rapid identification of chemical leads was demostrated by the discovery of (1) with micromolar affinity for the NK₃ receptor and (2), a weak inhibitor of the matrix metalloprotease MMP-1.     

Elucidation of discontinuous linear determinants in peptides.

Synthetic peptides, made by the method of simultaneous multiple peptide synthesis, were coupled to the protein carrier keyhole limpet hemocyanin and used to raise mAb. Omission and substitution analogs of the original peptides were tested by ELISA to characterize their reactivity with the respective mAb. Linear antigenic determinants were located for 18 different peptides by using omission analogs. The length of the antigenic determinants ranged from 2 to 8 residues, with an average of 6 residues. The three aromatic amino acids, phenylalanine, tryptophan, and tyrosine, the charged hydrophilic amino acids, aspartic acid and lysine, and the neutral amino acid alanine were found to occur most often in the determinant region of the peptides tested, whereas asparagine, cysteine, and histidine occurred the least often. Alanine substitution analogs provided more information than omission analogs by enabling the determination of which side chain groups of the antigenic determinant residues were not critical for binding to the mAb. Detailed, "fingerprint" information about the interaction of the peptide, GASPYPNLSNQQT, and its mAb was obtained by synthesizing a complete series of analogs with individual substitutions for each position of the antigenic determinant, PYPNLS, with the 19 other amino acids. These results suggest that, at the amino acid level, all antigenic determinants of synthetic peptides defined by mAb can be considered discontinuous linear determinants.     

Tartrazine: a potentially hazardous dye in Canadian drugs.

The literature was reviewed to determine the incidence of idiosyncratic reactions to tartrazine. From 4% to 14% of individuals with asthma or allergies or both and from 7% to 20% of persons who are sensitive to acetylsalicylic acid may react to this dye. The mechanism of such reactions is unknown. Pharmaceutical manufacturers and distributors were surveyed and a list was prepared of approximately 450 Canadian pharmaceuticals that contain tartrazine. The 53 pharmaceutical and manufacturers and distributors whose drug products do not contain this dye were also listed. It is recommended that information concerning the tartrazine content of drugs be included on package labels.     

Point mutations destabilizing a precursor protein enhance its post-translational import into mitochondria.

In order to study the role of protein unfolding during post-translational protein import into mitochondria, we destabilized the structure of a mitochondrial precursor protein by site-directed mutagenesis. The precursor consisted of the first 16 residues of the yeast cytochrome oxidase subunit IV precursor fused to mouse dihydrofolate reductase. Labilization of the folded precursor structure was monitored by increased susceptibility to protease and diminished ability of methotrexate to block import of the precursor into isolated yeast mitochondria. On comparing the original precursor with two mutant forms that were destabilized to different degrees, increased labilization correlated with an increased rate and efficiency of import into mitochondria. This supports the view that the precursor must unfold in order to enter the mitochondria.     

An overview of phytoremediation as a potentially promising technology for environmental pollution control

Phytoremediation is the use of plants for the removal of pollutants from contaminated soil or water. Phytoremediation is an environmentally friendly and cost-effective alternative to current remediation technologies. This review article outlines general aspects of phytoremediation, along with discussions about its advantages and limitations. It further reviews various phytoremediation processes in detail: phytoextraction, rhizofiltration, phytostabilization, phytodegradation, and phytovolatilization. Unlike previous review articles available in various journals, this paper presents a more comprehensive view of this issue, and deals with a much wider range of its applications to environmental pollution control. These include the treatment of wastewaters, removal of heavy metals and metalloids (e.g. lead and arsenic), phytoremediation of organic pollutants, such as 2,4,6-trinitrotoluene (TNT) and polychlorinated biphenyls (PCBs), and cleanup of soil and water contaminated with radionuclides, such as cesium (¹³⁷Cs) and strontium (⁹⁰Sr). This paper also describes recent developments of transgenic plants for improving phytoremediation. Along the way, the present status of phytoremediation research in Korea is briefly introduced. Finally, the article concludes with suggestions for future research.