Biomedical subjects

E Morag

Publications and source records attributed to E Morag.

At least 19 recordsLinked to original sources

PubMed2004

Production and purification of melanoma gp100 antigen and polyclonal antibodies.

gp100 is a melanoma-associated antigen found to carry immunogenic epitopes that can induce a CTL response against tumor cells. Production and purification of large quantities of this polypeptide may be important in the context of diagnosis and vaccinating against melanoma. To overcome the hydrophobic nature of gp100, we cloned and expressed only a part of the protein, and obtained a hydrophilic recombinant polypeptide (HR-gp100) that contained most of the immunogenic peptides. High yield was achieved in an Escherichia coli expression system. The protein was purified by AKTA Prime using anionic-columns. Polyclonal antibodies developed in chicken against HR-gp100 were efficient at detecting gp100 in melanoma cells, as determined by Western blot analysis and by immunohistochemistry. HR-gp100 can be used to develop a vaccine against melanoma. Antibodies to HR-gp100 may be used to detect tumors of melanocytic origin or to determine the level of gp100 expression in tumors prior to immunotherapy with the protein or one of its peptides.

Amino Acid Sequence↗

PubMed2001

Clinical competence assessment in radiology: introduction of an objective structured clinical examination in the medical school curriculum.

RATIONALE AND OBJECTIVES: Traditional oral and written examinations can be limited in predicting future clinical performance. Therefore Objective Structured Clinical Examinations (OSCEs) have been introduced in other specialties. The authors assessed their value in radiology. MATERIALS AND METHODS: The study includes 122 Harvard medical students who undertook 1-month compulsory clerkships at one of three hospitals (A, B, or C) in their 3rd and 4th year and a compulsory OSCE in their 4th year. The OSCE was constructed from five cases. Each had eight or nine standardized questions designed to test, within a set time, the perception of essential findings, their interpretation, and clinical judgment (maximum possible score, 100). Clerkship grades were high honors (score of 3), honors (score of 2), satisfactory (score of 1), and fail (score of 0). Predictors of OSCE scores-clerkship grade and affiliated hospital-were modeled as linear functions. Time elapsed between clerkship and OSCE was modeled as a nonlinear function. RESULTS: Although there was a positive relation between clerkship grade and OSCE grade, it accounted for an increase of only 5.7% in OSCE score per clerkship grade and did not predict performance of individual students. Students who trained in hospital B showed significantly higher OSCE grades. OSCE scores were highest when the examination was taken 8 months after the clerkship. CONCLUSION: The OSCE may be useful to uncover deficits in individuals and groups beyond the ones detected with traditional clerkship evaluations and provide guidance for remediation. The improved performance after additional clinical exposure suggests that the OSCE may be well suited to test the integration of radiologic and clinical knowledge.

Clinical Clerkship↗

PubMed2001

Nonproteolytic cleavage of aspartyl proline bonds in the cellulosomal scaffoldin subunit from Clostridium thermocellum.

Previous work from our group [Morage (Morgenstern), E., Bayer, E. A., and Lamed, R. (1991), Appl. Biochem. Biotechnol. 30, 129-136] has demonstrated an anomalous electrophoretic mobility pattern for scaffoldin, the 210-kDa cellulosome-integrating subunit of Clostridium thermocellum. Subsequent evidence [Morag, E., Bayer, E. A., and Lamed, R. (1992), Appl. Biochem. Biotechnol. 33, 205-217] indicated that the effect could be attributed to a nonproteolytic fragmentation of the subunit into a defined series of lower-molecular-weight bands. In the present work, a recombinant segment of the scaffoldin subunit was employed to determine the site(s) of bond breakage. An Asp-Pro sequence within the cohesin domain was identified to be the sensitive peptide bond. This sequence appears quite frequently in the large cellulosomal proteins, and the labile bond may be related to an as yet undescribed physiological role in the hydrolysis of cellulose by cellulosomes.

Amino Acid Sequence↗

PubMed2000

Abnormalities in muscle function during gait in relation to the level of lumbar disc herniation.

STUDY DESIGN: A comparison between gait in patients undergoing surgery for L4 and L5 lumbar disc herniations and that in an age- and weight-matched control group. OBJECTIVES: To study whether changes in the moments produced at the ankle and knee joints during walking reflect the neurologic level of a herniated nucleus pulposus. SUMMARY OF BACKGROUND DATA: Lumbar herniated discs often cause muscle weakness, reduced motor function, and change in walking capacity. The specific effects of a disc herniation on muscle function during gait is poorly documented. METHODS: Conventional physical examination and kinetic analysis of gait were performed on 16 patients who subsequently underwent surgery for herniated discs (eight with L4-L5 and eight with L5-S1 disc herniations) and 16 healthy control subjects. The three components of the external moment at the ankle and knee were computed. The peak magnitudes of specific components of the external moments were compared with those of the control group. RESULTS: Reduced external ankle plantar flexion moment, indicating a decreased function of the ankle dorsiflexors, was found in patients with herniated nucleus pulposus of both L4-L5 and L5-S1. Reduced external ankle dorsiflexion moment, indicating a decreased function of the ankle plantar flexors, was found only in patients with a lesion to the L5-S1 disc, but not in those with herniations at L4-L5. CONCLUSIONS: Preoperative gait analysis identified functional deficits of the muscles about the ankle and foot that relate to the level of the herniation. Kinetic measurements can assist in understanding the functional limitations associated with specific levels of a herniation.

Adult↗

PubMed1999

Structural and functional predictors of regional peak pressures under the foot during walking.

The objective of this study was to identify structural and functional factors which are predictors of peak pressure underneath the human foot during walking. Peak plantar pressure during walking and eight data sets of structural and functional measures were collected on 55 asymptomatic subjects between 20 and 70 yr. A best subset regression approach was used to establish models which predicted peak regional pressure under the foot. Potential predictor variables were chosen from physical characteristics, anthropometric data, passive range of motion (PROM), measurements from standardized weight bearing foot radiographs, mechanical properties of the plantar soft tissue, stride parameters, foot motion in 3D, and EMG during walking. Peak pressure values under the rearfoot, midfoot, MTH1, and hallux were measured. Heel pressure was a function of linear kinematics, longitudinal arch structure, thickness of plantar soft tissue, and age. Midfoot pressure prediction was dominated by arch structure, while MTH1 pressure was a function of radiographic measurements, talo-crural joint motion, and gastrocnemius activity. Hallux pressure was a function of structural measures and MTP1 joint motion. Foot structure and function predicted only approximately 50% of the variance in peak pressure, although the relative contributions in different anatomical regions varied dramatically. Structure was dominant in predicting peak pressure under the midfoot and MTH1, while both structure and function were important at the heel and hallux. The predictive models developed in this study give insight into potential etiological factors associated with elevated plantar pressure. They also provide direction for future studies designed to reduce elevated pressure in "at-risk" patients.

Adult↗

PubMed1998

Does a single control mechanism exist for both forward and backward walking?

It has been proposed that the highly reproducible forward walking (FW) locomotor pattern is generated by a central neuronal program or central pattern generator (CPG) which provides the underlying mechanism which produces the coordinated walking movement. The purpose of this study was to quantify the differences in the muscular activation patterns during FW and backward walking (BW) at a constant step frequency and to determine if common features exist across both locomotor conditions. The hypothesis was that FW and BW are both mediated by the same CPG; therefore, only small modifications in the CPG are required in order to produce the different characteristics of each walking mode. The results noted kinematically reversed patterns at the hip and ankle joints between FW and BW. The knee joint movement pattern was similar between conditions, however, a phase shift of 14.3% of the gait cycle occurred. An approximately 25% phase shift in the muscle activation patterns existed between FW and BW in four of the six muscles studied. Additionally, a pattern recognition technique was applied to the combined EMG signals to determine the minimum number of features required to generate the measured muscular output. Only two main features were necessary to produce the EMG patterns for both the FW and BW condition. The main features in FW were more consistent than noted in BW. The results support the notion that a single spinal mechanism such as a CPG with two main features appears to be in control during both FW and BW. Copyright 1998 Elsevier Science B.V. All rights reserved

Journal Article↗

PubMed1997

A cohesin domain from Clostridium thermocellum: the crystal structure provides new insights into cellulosome assembly.

BACKGROUND: The scaffoldin component of the cellulolytic bacterium Clostridium thermocellum is a non-hydrolytic protein which organizes the hydrolytic enzymes in a large complex, called the cellulosome. Scaffoldin comprises a series of functional domains, amongst which is a single cellulose-binding domain and nine cohesin domains which are responsible for integrating the individual enzymatic subunits into the complex. The cohesin domains are highly conserved in their primary amino acid sequences. These domains interact with a complementary domain, termed the dockerin domain, one of which is located on each enzymatic subunit. The cohesin-dockerin interaction is the crucial interaction for complex formation in the cellulosome. The determination of structural information about the cohesin domain will provide insights into cellulosome assembly and activity. RESULTS: We have determined the three-dimensional crystal structure of one of the cohesin domains from C. thermocellum (cohesin 2) at 2.15 A resolution. The domain forms a nine-stranded beta sandwich with a jelly-roll topology, somewhat similar to the fold displayed by its neighboring cellulose-binding domain. CONCLUSIONS: The compact nature of the cohesin structure and its lack of a defined binding pocket suggests that binding between the cohesin and dockerin domains is characterized by interactions between exposed surface residues. As the cohesin-dockerin interaction appears to be rather nonselective, the binding face would presumably be characterized by surface residues which exhibit both intraspecies conservation and interspecies dissimilarity. Within the same species, unconserved surface residues may reflect the position of a given cohesin domain within the scaffoldin subunit, its orientation and interactions with neighboring domains.

Amino Acid Sequence↗

PubMed1997

Crystallization and preliminary X-ray analysis of a cohesin domain of the cellulosome from Clostridium thermocellum.

Recombinant cohesin-2, a unique type of protein-recognition domain from the cellulosome of Clostridium thermocellum, has been crystallized by the hanging-drop vapor-diffusion method. The crystals are monoclinic, space group C2 with unit-cell dimensions a = 79.91, b = 47.86, c = 51.13 A, beta = 126.77 degrees. There is most likely to be one molecule per asymmetric unit, corresponding to a packing density of 2.16 A(3) Da(-1). The crystals diffract to beyond 2.3 A on a conventional laboratory rotating-anode source.

Journal Article↗

PubMed1997

The relationship of static foot structure to dynamic foot function.

Many theories have been advanced concerning the relationship between structure and function in the human foot, yet few of these theories have been subjected to quantitative examination. In this study, foot structure was characterized by 27 measurements taken from standardized lateral and dorsi-plantar weight-bearing plain radiographs of 50 healthy adult subjects. Regional plantar pressure distribution data collected from the same feet were chosen as the functional measures. A stepwise regression analysis was performed to (1) explore what portion of the variance in peak plantar pressure during walking can be explained by the radiographic measurements, and (2) identify structural characteristics of the foot which are significant predictors of peak plantar pressure under the heel and the first metatarsal head (MTH1). Most of the radiographic measurements were highly reliable. However, only 31 and 38% of the variance in peak plantar pressure at the heel and MTH1, respectively, could be explained using multiple regression analyses with the radiographic measurements as independent variables. Among the structural predictors that were identified, soft tissue thickness (e.g. calcaneus or sesamoid heights), and arch-related measurements were the strongest predictors of plantar pressure under both the heel and the first metatarsal head. We conclude that, in normal subjects, only about 35% of the variance in dynamic plantar pressure can be explained by the measurements of foot structure derived from radiographs. This implies that the dynamics of gait are likely to exert the major influences on plantar pressure during walking.

Adult↗

PubMed1997

Structural and functional aspects of the diabetic foot.

INTRODUCTION:: It has been well documented that, on average, patients with diabetes mellitus (DM) have higher plantar pressures than persons without DM, and there are several hypotheses why this may be -- many focused on the role of peripheral neuropathy. The purposes of this study were: (A) To identify and quantify structural differences between age-matched diabetic and non diabetic subjects, and (B) to develop predictive equations for peak pressure in these same patients. METHODS:: Standardized lateral and dorsi-plantar weight bearing plain radiographs of the right foot and ankle of 50 symptom free (NDM), 32 diabetic with no signs of neuropathy (NNP), and 73 diabetic neuropathic (NP) subjects were taken by a single radiographer. Twenty six angular and linear measurements were then made from the films. Plantar pressure distribution from three first step walking trials were collected and peak pressures at 10 discrete sites were determined for each step, and averaged. A one-way ANOVA with Tukey post hoc tests was conducted to test for differences between the means of each of the 26 radiographic measurements at the 0.01 significant level. Regression analysis was also used to predict the ln(pressure) under the heel, midfoot, mth1, mth2, and mth5 regions from the radiographic measurements, range of motion at the talocrural and first metatarsophalangeal joints and weight, for each of the 3 groups independently. RESULTS:: Significant differences were found among the three groups in 5 of the 26 radiographic measurements (Table 1). These included the thickness of the first, second and third metatarsals from the AP view (MT1, MT2 and MT3 -- generally > in NP), and the sesamoid height (NP > others) and fifth metatarsal head height (NDM < others). In addition, non significant trends were found in 4 other radiographic measurements. Regression analysis identified groups of up to five[Table: see text] predictors which accounted for between 27% and 62% of the variance in peak plantar pressure in different subject groups and foot regions. DISCUSSION AND CONCLUSIONS:: We conclude that objective differences in foot structure are present in diabetic neuropathic subjects, and that DM per se is apparently not a factor in these differences. We also concluded that static foot structure accounts for anywhere between approximately 30% to 60% of the variance in regional peak pressure, depending on the region investigated. In most cases predictions were higher in the midfoot and forefoot.

Journal Article↗

PubMed1997

Species-specificity of the cohesin-dockerin interaction between Clostridium thermocellum and Clostridium cellulolyticum: prediction of specificity determinants of the dockerin domain.

The cross-species specificity of the cohesin-dockerin interaction, which defines the incorporation of the enzymatic subunits into the cellulosome complex, has been investigated. Cohesin-containing segments from the cellulosomes of two different species, Clostridium thermocellum and Clostridium cellulolyticum, were allowed to interact with cellulosomal (dockerin-containing) enzymes from each species. In both cases, the cohesin domain of one bacterium interacted with enzymes from its own cellulosome in a calcium-dependent manner, but the same cohesin failed to recognize enzymes from the other species. Thus, in the case of these two bacteria, the cohesin-dockerin interaction seems to be species-specific. Based on intra- and cross-species sequence comparisons among the different dockerins together with their known specificities, we tender a prediction as to the amino-acid residues critical to recognition of the cohesins. The suspected residues were narrowed down to only four, which comprise a repeated pair located within the calcium-binding motif of two duplicated sequences, characteristic of the dockerin domain. According to the proposed model, these four residues do not participate in the binding of calcium per se; instead, they appear to serve as recognition codes in promoting interaction with the cohesin surface.

Amino Acid Sequence↗

PubMed1996

Immobilized nitro-avidin and nitro-streptavidin as reusable affinity matrices for application in avidin-biotin technology.

Chemically modified forms of egg-white avidin and bacterial streptavidin (termed nitro-avidin and nitro-streptavidin, respectively), in which the binding-site tyrosine was nitrated, were used for several biotechnological applications. The fundamental difference between nitro-avidin and the native protein is that interaction of the modified protein with biotin can be reversed under relatively mild conditions. Consequently, nitro-avidin affinity columns or immobilizing matrices can be reused. Three examples are given to demonstrate the possible uses of such columns: (a) biotinylated protein A was attached to a nitro-avidin affinity column, and immunoglobulin was purified directly from whole rabbit serum; (b) biotinylated transferrin was attached to a nitro-streptavidin column, and anti-transferrin was isolated directly from rabbit anti-serum; and (c) biotinylated beta-glucosidase was immobilized onto a nitro-avidin column and used as an enzyme reactor. In each example, the immobilized biotinylated probe could be released selectively from the column and recovered following its utilization. Reusable nitro-avidin thus provides an easy and attractive reversible form of avidin and thereby serves to expand the versatility of avidin-biotin technology.

Animals↗

PubMed1996

Recovery of high-affinity phage from a nitrostreptavidin matrix in phage-display technology.

A novel approach for the selection of high-affinity phage from phage-peptide libraries is described. The methodology employs a chemically modified form of streptavidin, termed nitrostreptavidin, which exhibits a reversible attraction for biotin. The new approach emulates conventional procedures in that a biotinylated probe, in this case biotinylated alpha-bungarotoxin, is attached to an immobilized streptavidin matrix. The phage library is introduced, and interacting phage particles are released under conventional acidic conditions (pH 2.2). At this stage, the primary peptide sequences characterizing the released phage are found to be identical with those previously known to interact with the toxin. However, other phage particles, which presumably interact more strongly than those released by acid, remain attached to the immobilized toxin. These can be released by virtue of the reversible biotin-binding properties of nitrostreptavidin. For this purpose, alkaline solutions (pH 10) or free biotin can be used. Using this approach, phage particles that recognize alpha-bungarotoxin were isolated; their peptide sequences were found to be similar to, but clearly distinct from, those collected by conventional acid elution. The affinity of the isolated phage was dramatically higher than that of phage obtained by the conventional methodology. In contrast, their synthetically prepared 15-mer peptides actually exhibited a lower affinity for the toxin than that shown by peptides prepared on the basis of the sequence obtained from conventional acid-eluted phage. This apparent discrepancy can be explained by an altered conformational state of the peptides in solution, compared to the epitopes expressed in situ on the phage surface.

Adsorption↗

PubMed1996

Expression, purification and crystallization of a cohesin domain from the cellulosome of Clostridium thermocellum.

The cellulosome of the cellulolytic bacterium, Clostridium thermocellum, is a multi-enzyme complex in which the enzymatic (cellulolytic) subunits are attached to a unique nonhydrolytic subunit called scaffoldin. The attachment is mediated by two mutually interacting domains: namely multiple cohesin domains on the scaffoldin subunit and a dockerin domain on each of the enzymatic subunits. Knowledge of the three-dimensional structure of each of the interacting components would be critical to a better understanding of the cohesin-dockerin interaction at the molecular level. In this report, we describe the purification of one of the nine cohesin domains of the scaffoldin subunit from C. thermocellum. A DNA segment containing the cohesin 2 sequence was fused to a hexa-histidine tag, and the resultant construct was expressed in Escherichia coli. The expressed peptide was efficiently isolated by metal-chelate affinity chromatography. The purified recombinant form of the cohesin was crystallized pending determination of its structure.

Amino Acid Sequence↗

PubMed1996

Crystal structure of a bacterial family-III cellulose-binding domain: a general mechanism for attachment to cellulose.

The crystal structure of a family-III cellulose-binding domain (CBD) from the cellulosomal scaffoldin subunit of Clostridium thermocellum has been determined at 1.75 A resolution. The protein forms a nine-stranded beta sandwich with a jelly roll topology and binds a calcium ion. conserved, surface-exposed residues map into two defined surfaces located on opposite sides of the molecule. One of these faces is dominated by a planar linear strip of aromatic and polar residues which are proposed to interact with crystalline cellulose. The other conserved residues are contained in a shallow groove, the function of which is currently unknown, and which has not been observed previously in other families of CBDs. On the basis of modeling studies combined with comparisons of recently determined NMR structures for other CBDs, a general model for the binding of CBDs to cellulose is presented. Although the proposed binding of the CBD to cellulose is essentially a surface interaction, specific types and combinations of amino acids appear to interact selectively with glucose moieties positioned on three adjacent chains of the cellulose surface. The major interaction is characterized by the planar strip of aromatic residues, which align along one of the chains. In addition, polar amino acid residues are proposed to anchor the CBD molecule to two other adjacent chains of crystalline cellulose.

Amino Acid Sequence↗

PubMed1996

Reversibility of biotin-binding by selective modification of tyrosine in avidin.

The tight interaction between the vitamin biotin and the protein avidin is so strong (Ka approximately 10(15) M-1) that conditions which are usually sufficient for protein denaturation fail to dissociate the avidin-biotin complex. In order to form a reversible interaction between the two biomolecules, we have modified the binding-site tyrosine by nitration, thus reducing the pKa of the phenol group which forms a crucial hydrogen bond with the ureido group of biotin. At relatively low pH values (4-5), the resultant modified forms of avidin bind biotin with a very high association constant ( > 10(9) M-1). The modified avidins are thus capable of supporting stable, long-term binding of biotin or biotinylated macro-molecules. The latter molecules can be detached by increasing the pH of the medium or by introduction of excess levels of biotin at neutral pH. These findings demonstrate the importance of a single hydrogen bond for strong biotin binding. The new derivatives of avidin should be useful for applications whereby a reversible interaction between the four biotin-binding sites and biotin is desired, thus increasing the versatility of the avidin-biotin system for biotechnological application.

Avidin↗

PubMed1995

Evidence for a general role for non-catalytic thermostabilizing domains in xylanases from thermophilic bacteria.

A genomic library of Clostridium thermocellum DNA constructed in lambda ZAPII was screened for xylanase-expressing clones. Cross-hybridization experiments revealed a new xylanase gene isolated from the gene library, which was designated xyn Y. The encoded enzyme, xylanase Y (XYLY), displayed features characteristic of an endo-beta1,4-xylanase: the enzyme rapidly hydrolysed oat spelt, wheat and rye arabinoxylans and was active against methyl-umbelliferyl-beta-D-cellobioside, but did not hydrolyse any cellulosic substrates. The pH and temperature optima of the enzyme were 6.8 and 75 degrees C respectively, and the recombinant XYLY, expressed by Escherichia coli had a maximum Mr of 116000. The nucleotide sequence of xyn Y contained an open reading frame of 3228 bp encoding a protein of predicted Mr 120 105. The encoded enzyme contained a typical N-terminal 26-residue signal peptide, followed by a 164 amino acid sequence, designated domain A, that was not essential for catalytic activity. Downstream of domain A was a 351-residue xylanase Family F catalytic domain, followed by a 180-residue sequence that exhibited 28% sequence identity with a thermostable domain of Thermoanaerobacterium saccharolyticum xylanase A. The C-terminal portion of XYLY comprised the 23-residue duplicated docking sequence found in all other C. thermocellum plant cell wall hydrolases that are constituents of the bacterium's multienzyme complex, termed the cellulosome, followed by a 286-residue domain which exhibited 32% sequence identity with the N-terminal region of C. thermocellum xylanase Z. The enzyme did not contain linker sequences found in other C. thermocellum plant cell wall hydrolases. Analysis of truncated forms of XYLY and hybrid proteins, comprising segments of XYLY fused to the E. coli maltose binding domain, confirmed that XYLY contained a central catalytic domain and an adjacent thermostable domain. The C-terminal domain did not bind to cellulose or xylan. Western blot analysis using antiserum raised against XYLY showed that the xylanase was located in the cellulosome and did not appear to be extensively glycosylated. The non-catalytic domains of XYLY are discussed in relation to the general stability of thermophilic xylanases.

Amino Acid Sequence↗

PubMed1995

Expression, purification and subunit-binding properties of cohesins 2 and 3 of the Clostridium thermocellum cellulosome.

The enzymatic subunits of the cellulosome of Clostridium thermocellum are integrated into the complex by a major non-catalytic polypeptide, called scaffoldin. Its numerous functional domains include a single cellulose-binding domain (CBD) and nine subunit-binding domains, or cohesin domains. Two of the cohesin domains, together with the adjacent CBD, have been cloned and expressed in Escherichia coli, and the recombinant constructs were purified by affinity chromatography on a cellulosic matrix. Both cohesin domains, which differ by about 30% in their primary structure, showed a similar binding profile to the cellulosomal subunits. Calcium ions enhanced dramatically this binding. Under the conditions of the assay, only one major catalytic subunit of the cellulosome failed to bind to either cohesin domain. The results indicate a lack of selectivity in the binding of cohesin domains to the catalytic subunits and also suggest that additional mechanisms may be involved in cellulosome assembly.

Amino Acid Sequence↗