Unfolding and refolding of the N-terminal fragments of staphylococcal nuclease R in guanidine hydrochloride

The conformational and activity changes of a family of peptide fragments of staphylococcal nuclease R, which extend from residues -6 to 102, -6 to 110, -6 to 121, -6 to 135, and -6 to 141, during unfolding and refolding in different concentrations of guanidine hydrochloride have been studied. The studies indicate that the conformational stability in guanidine hydrochloride solution of the N-terminal fragment increases with increasing chain length, and that interaction and recognition between amino acid residues which are related to formation of the native conformation also increase with growth of the peptide chain, but such interaction becomes effective only when the polypeptide chain reaches a certain length. The changes in conformation and catalytic activity of the N-terminal fragments during unfolding and refolding demonstrate that conformational adjustments are necessary during chain elongation to generate the native conformation of a biologically active protein.     

Folding of SNase R begins early during synthesis: the conformational feature of two short N-terminal fragments of staphylococcal nuclease R

The objective was to determine why some people who are involved in minor motor vehicle accidents, without loss of consciousness, have persisting headaches and neckache, and to suggest management of these symptoms. Between 1954 and 1994, over 4400 cases were referred for medico-legal opinions. A group has been selected for discussion. During the period 1954-1966, 414 cases following closed head injuries were seen with varying periods of post traumatic amnesia (PTA) from nil to greater than 72 h. The average time between the accident and the examination was 21 months. The shortest period was 3 months and the longest 7 years. The age at the time of the accident varied from 2.5 to 72 years. The largest group fell between the ages of 20 and 40 years. The main complaints were headache, giddiness, loss of concentration and poor memory. 380 were reviewed by questionnaire after settlement of the case. 112 cases of extension/flexion injuries of the neck were seen between 1985 and 1989 and their symptoms and resolution were compared with 50 cases seen over the same period following significant head or neck injury. The results showed that the more severe the head or neck injury, the less likely were the cases to suffer symptoms of post-traumatic headaches or persisting neck symptoms. In conclusion, while 70% of minor head and neck injuries settle within a few weeks of a motor vehicle accident, about 30% continue to complain of headaches and/or neck pain. The prolonged management, extensive physiotherapy and slow court settlement lead to excessive introspection and prolongation of symptoms.     

Reduction of renal uptake of monoclonal antibody fragments by amino acid infusion

The renal uptake of radiolabeled antibody fragments and peptides presents a problem in radioimmunodetection and therapy, compromising lesion sensitivity, especially with intracellularly-retained isotopes. Previously, we showed that cationic amino acids and their derivatives are capable of significantly reducing kidney uptake in animals. We report our initial clinical results of successful renal uptake reduction in five patients who underwent cancer radioimmunodetection with 99mTc-anti-CEA Fab' fragments. METHODS: The patients were infused with two liters of a commercially-available nutritive amino acid solution (containing approximately 2.25 g/liter lysine-glutamate and 2.50 g/liter arginine), whereas 75 control patients received the same volume of saline (quantification of organ and tumor kinetics from conjugate whole-body views by ROI technique). RESULTS: The renal uptake in the amino acid group was significantly lower (p<0.05) than in the control group (11.1 +/- 2.0% injected dose versus 17.7 +/- 7.0% injected dose at 24 hr postinjection), whereas the uptake of all other organs remained unaffected. Gel filtration chromatography of the urine taken from amino-acid-treated patients showed that a significantly higher amount of excreted activity was bound to intact Fab' (53% of excreted activity) in contrast to only less than 10% in the control group. CONCLUSION: The renal uptake of monoclonal antibody fragments in patients can be reduced significantly by amino acid infusion, even at considerably lower doses than those that were safe and effective in animals. As was found in animals, the mechanism seems to rely on an inhibition of the re-absorption of tubularly-filtered proteins by the proximal tubule cells. These results encourage further clinical trials to lower the renal uptake experienced in radioimmunodetection, as well as in therapeutic trials with antibody fragments and peptides.     

A revised strategy for cloning antibody gene fragments in bacteria

Rhinoviruses and enteroviruses are the major members of the picornavirus genus that cause human disease. We compared the polymerase chain reaction and viral culture for the identification of picornaviruses in nasal aspirates from children during episodes of respiratory symptoms and when asymptomatic and from asymptomatic adults. One hundred eight children, aged 9 to 11 years, completed a year-long study. Within 24 to 48 h of a report of respiratory symptoms, a nasal aspirate was taken in the home. Nasal aspirates were also taken from 65 of the children and from 33 normal adults when they had been free of respiratory symptoms for at least 2 weeks. Picornaviruses were isolated by culture for three passages in Ohio HeLa cells in rolling tubes at 33 degrees C and pH 7.0. For the polymerase chain reaction, duplicate 50-microliters samples were amplified with conserved primers from the 5' noncoding region. Picornaviruses generated approximately 380-bp bands in agarose gel electrophoresis; the specificity of these bands was confirmed by filter hybridization with a conserved internal probe. Picornaviruses were isolated by culture in 47 (46 rhinoviruses) of 292 symptomatic episodes (16%), whereas the polymerase chain reaction identified picornavirus genomic material in 146 episodes (50%), including all but one of the culture-positive episodes. As for asymptomatic samples, eight (12%) children and two (4%) adults were positive by the polymerase chain reaction, whereas only one child's specimen was positive by culture. This polymerase chain reaction assay represents a clear advance in the identification of picornavirus infection, with a detection rate threefold greater than the virus culture method.     

Stability effects of increasing the hydrophobicity of solvent-exposed side chains in staphylococcal nuclease

A total of fifty single site surface phenylalanine substitution mutants have been made in the model protein staphylococcal nuclease. The fifty residues that were replaced with phenylalanine were chosen to give a broad sampling of solvent accessibility, secondary structure, and backbone conformations. The change in the stability of each mutant protein relative to wild type was measured by guanidine hydrochloride denaturation. These results were compared to previous results obtained when these same sites were substituted with an alanine and a glycine. By this means, changes in the stability due to the loss of interactions of the wild-type side chain can be separated from the effects of introducing the bulky, hydrophobic phenylalanine in these solvent-exposed positions. In general, our results agree with the conventional wisdom that placing a hydrophobic residue in a solvent-exposed position is destabilizing in most cases, but less destabilizing than most changes in the hydrophobic core of the protein. However, the degree to which a hydrophobic surface substitution destabilizes or stabilizes a globular protein is highly context-dependent, with some mutations being as destabilizing as those in the core. This indicates that steric and packing considerations are also important on the surface of a globular protein but generally are not as important as in the interior. No evidence for the widespread occurrence of the so-called reverse hydrophobic effect at solvent-exposed sites was found. In addition, this survey of numerous sites suggests that previous measurements of alpha-helix "propensities" often seriously underestimate the importance of the environment of the side chain.     

Stability studies of amino acid substitutions at tyrosine 27 of the staphylococcal nuclease beta-barrel

In order to help determine the extent to which side chain interactions within the staphylococcal nuclease beta-barrel affect its global stability, a full set of point mutants was generated for residue 27. Intrinsic tryptophan fluorescence was monitored during solvent denaturation with guanidine hydrochloride (GuHCl) and was used to calculate DeltaGH2O unfolding and m values for each mutant. In the wild type protein, residue 27 is a tyrosine which is at the first position of a type I' beta-turn, and which participates in both hydrophobic interactions and side chain to side chain hydrogen bonding. The hydrophobicity of the mutant residue was found to be the dominant factor in determining global protein stability within this series of nuclease mutants.     

Generation of rabbit monoclonal antibody fragments from a combinatorial phage display library and their production in the yeast Pichia pastoris

We have applied the combinatorial immunoglobulin library and phage display technologies to generate monoclonal rabbit single-chain Fv (scFv) antibody fragments specific for recombinant human leukemia inhibitory factor (rhLIF). The B cell immunoglobulin repertoire of an immunized rabbit was immortalized by the combinatorial cloning of the rearranged variable domains of light (VL) and heavy (VH) chains. Affinity selection of the library displaying the rabbit antibody domains on the phage surface resulted in the isolation of phage encoding scFv antibodies which specifically bind to the antigen. We utilized the methylotrophic yeast Pichia pastoris for high level secretion of soluble and functional scFv antibody fragment. More than 100 mg/L of pure and functional rabbit anti-rhLIF scFv antibody was obtained directly from the P. pastoris culture supernatant by one-step affinity chromatography.     

Human tryptase fibrinogenolysis is optimal at acidic pH and generates anticoagulant fragments in the presence of the anti-tryptase monoclonal antibody B12

Human tryptase is uniquely regulated by its association with heparin and resists inhibition by biological protease inhibitors. The effects of pH and B12, an IgG anti-tryptase mAb, on cleavage of the synthetic substrate tosyl-Gly-Pro-Lys-p-nitroanilide and of the biological substrate fibrinogen by tryptase were examined. Tosyl-Gly-Pro-Lys-pnitroanilide cleavage was optimal at neutral pH and was inhibited by the B12 mAb at acidic and neutral pH values. At pH 7.5, inhibition was reversible and noncompetitive. In contrast, the optimal pH for tryptase to cleave fibrinogen was acidic. B12 dramatically enhanced the rate and extent that tryptase cleaved all three fibrinogen subunits at pH 6.0 to 6.5, but inhibited these activities at neutral pH. Major fibrinogen cleavage fragments generated at acidic pH by the B12:tryptase complex were identical with those made by plasmin. Thus, at acid pH, tryptase alone destroyed the ability of fibrinogen to clot, while the B12:tryptase complex increased the rate of fibrinogenolysis and also generated the anticoagulant, fragment D. The acidic pH optimum for tryptase fibrinogenolysis may direct this activity to tissue sites of inflammation. A putative biological equivalent to B12 would limit tryptase fibrinogenolytic activity at sites of neutral pH, such as blood, but would augment activity at acidic sites.     

A monoclonal antibody affects chromophore apoprotein interactions of phytochrome A

A monoclonal antibody designated Mep-1 was raised against phytochrome A from pea (Pisum sativum L.). The binding of this antibody (class IgG1) to partially degraded phytochrome (114 kDa) caused a considerable increase in the far-red peak at the red-light-induced stationary state. The effect reached a plateau value when the antibody and phytochrome were present in approximately equimolar amounts. The dark transformation of the far-red-light-absorbing form to the red-light-absorbing form of the 114 kDa phytochrome was inhibited by the addition of the antibody. However, binding of the antibody to the undegraded 121 kDa phytochrome had no effects on the spectrum of the red-light-induced steady state. The site at which the antibody bound to phytochrome was determined to be between amino acid residues 256 and 383 of pea phytochrome A. This is the first report of a monoclonal antibody that enhances the far-red absorption of phytochrome in the red-light-induced photostationary state.     

Specific detection of myeloma plasma cells using anti-idiotypic single chain antibody fragments selected from a phage display library

OBJECTIVES: The authors examined how the courts have responded to public and private insurers' use of medical appropriateness criteria to establish coverage and payment policies. METHODS: A structured review of all federal and state court health insurance cases decided between 1960 and June 1994 that involved a dispute involving medical appropriateness was performed. A total of 3,215 published court decisions were analyzed, of which 203 met the criteria of relevance and 124 explicitly mentioned medical appropriateness criteria. The main outcome variable was whether the court ordered the insurer to provide coverage. RESULTS: In 185 cases, a definitive decision was rendered, and the insurer was required to pay in 57% of the decisions. Whether the insurer relied on an assessment or not, whether the assessment process was formal or informal, and who conducted the assessment did not appear to influence courts' decisions, nor did the specificity of the coverage exclusion. Significant predictors of courts ordering coverage were court jurisdiction, contract language assigning discretion to the insurer, severity of patient's condition, and whether the treatment appeared to work for the particular patient. CONCLUSIONS: For practice guidelines to be accepted by the courts, it is more important to focus on how insurance contracts are written than on how medical assessments are performed.     

An N-terminal region of LukF of staphylococcal leukocidin/gamma-hemolysin crucial for the biological activity of the toxin

The two staphylococcal bi-component toxins, leukocidin and gamma-hemolysin share LukF [Kamio et al, FEBS Lett., 321, 15-18 (1993)]. This report identifies the pivotal amino acid residues in the N-terminal region of LukF for the leukocytolytic and hemolytic activities in the presence of LukS and HIg2, respectively, measuring the toxin activity of a series of LukF mutants with truncated N-terminals. The data obtained showed that the LukF mutant TF21, lacking 20 amino acid residues at the N-terminus of LukF, failed to have any hemolytic activity and had less 10% leukocytolytic activity than that of the intact LukF, while 16-residue truncations retained both toxin activities without loss. The LukF mutants lacking 18- through 19-residue segments from the N-terminus showed low toxin activity on both target cells. All mutants having no toxin activity were also not capable of binding to the human erythrocytes. It can thus be concluded that the 3-residue segment, L18Y19K20 of LukF is crucial for the biological activity of the toxin.     

A protein-specific monoclonal antibody to rat liver beta 1-->4 galactosyltransferase and its application to immunohistochemistry

BACKGROUND: The co-occurrence of panic disorder and major depression in the same individual is common. A question to be answered is whether the comorbid disorder is a distinct one or may resemble one or other disorder. In this paper we examine whether the comorbid disorder is a distinct condition. METHOD: We examined the symptom profiles and rates of comorbidity of panic attacks and DIS/DSM-III major depressive disorder in a population-based sample from four sites of the National Institute of Mental Health (NIMH) Epidemiologic Catchment Area Program (n = 12,668). RESULTS: The co-occurrence of panic attacks and major depression over the lifetime was 11 times higher than expected by chance (OR = 11.4, 95% CI 9.5 to 13.6). Subjects with both panic and depression had worse symptoms than those who had only one disorder. However, the pattern of symptoms was remarkably similar, after overall severity was taken into account. Depressive symptoms associated with more severe forms of depression (e.g. guilt, suicidal thoughts or attempts, and motor disturbance) were more frequent in the comorbid group. CONCLUSIONS: These findings may indicate a worse severity when the two disorders occur in the same individual.     

A monoclonal antibody directed against the non-toxic subunit of a dimeric phospholipase A2 neurotoxin, crotoxin, neutralizes its toxicity

Cancer patients undergoing radiotherapy frequently report fatigue. However, knowledge of the importance of fatigue for these patients and of the factors associated with their fatigue is limited. The aim of the current investigation was to gain more insight into fatigue as related to radiotherapy by answering the following questions. First, how is the experience of fatigue best described? Secondly, to what extent is fatigue related to sociodemographic, medical (including treatment), physical and psychological factors? Finally, is it possible to predict which patients will suffer from fatigue after completion of radiotherapy? Patients with different types of cancer receiving radiotherapy with curative intent (n = 250) were interviewed before and within 2 weeks of completion of radiotherapy. During treatment, patients rated their fatigue at 2-weekly intervals. Results indicate a gradual increase in fatigue over the period of radiotherapy and a decrease after completion of treatment. Fatigue scores obtained after radiotherapy were only slightly, although significantly, higher than pretreatment scores. After treatment, 46% of the patients reported fatigue among the three symptoms that caused them most distress. Significant associations were found between post-treatment fatigue and diagnosis, physical distress, functional disability, quality of sleep, psychological distress and depression. No association was found between fatigue and treatment or personality characteristics. Multivariate regression analysis demonstrated that the intensity of pretreatment fatigue was the best predictor of fatigue after treatment. In view of this finding, a regression analysis was performed to gain more insight into the variables predicting pretreatment fatigue. The degree of functional disability and impaired quality of sleep were found to explain 38% of the variance in fatigue before starting radiotherapy. Fatigue in disease-free patients 9 months after treatment is described in paper (B) in this issue.     

Cancer imaging with radiolabeled antibodies: new advances with technetium-99m-labeled monoclonal antibody Fab' fragments, especially CEA-Scan and prospects for therapy

The use of radiolabeled anticancer antibodies to detect cancer sites by external scintigraphy has had a relatively long history. With the advent of monoclonal antibodies (MAbs), which precluded the need for purifying the antibodies by laborious purification steps, there was a surge of interest and efforts to develop these reagents for both imaging and therapy applications (1). Today, many thousands of patients have received different forms and doses of MAbs for various purposes, and four MAb-based products have been licensed for manufacture and sale in the U.S. (2,3). This article describes the most recent MAb product to be approved in the U.S. for colorectal cancer imaging, including discussions of using this agent and its therapeutic counterpart in several cancer types.     

Incorporation of tryptophan analogues into staphylococcal nuclease, its V66W mutant, and Delta 137-149 fragment: spectroscopic studies

The tryptophan analogues, 5-hydroxytryptophan, 7-azatryptophan, 4-fluorotryptophan, 5-fluorotryptophan, and 6-fluorotryptophan, have been biosynthetically incorporated into Staphylococcal nuclease, its V66W mutant, and the Delta 137-149 fragment of the latter mutant. The guanidine-HCl induced unfolding and thermal unfolding of these proteins were studied to characterize the effect of incorporation of these tryptophan analogues on the thermodynamic stability of the proteins. The three proteins have tryptophan residues at positions 140 (in wild type) and 66 (in the Delta 137-149 fragment of V66W) and at both positions (in V66W). The unfolding data show that 5-hydroxytryptophan does not perturb the stability of wild-type nuclease, but it destabilizes the fragment and causes the V66W mutant to unfold in a more cooperative manner. 7-Azatryptophan is found to destabilize all three proteins. 4-Fluorotryptophan is slightly stabilizing of the three proteins, but the other two fluorotryptophans do not alter the stability of the proteins.     

Interactions in nonnative and truncated forms of staphylococcal nuclease as indicated by mutational free energy changes

Several mixed disulfide variants of staphylococcal nuclease have been produced by disulfide bond formation between nuclease V23C and methane, ethane, 1-propane, 1-n-butane, and 1-n-pentane thiols. Although CD spectroscopy shows that the native state is largely unperturbed, the stability toward urea-induced unfolding is highly dependent on the nature of the group at this position, with the methyl disulfide protein being the most stable. The variant produced by modification with iodoacetic acid, however, gives a CD spectrum indicative of an unfolded polypeptide. Thiol-disulfide exchange equilibrium constants between nuclease V23C and 2-hydroxyethyl disulfide have been measured as a function of urea concentration. Because thiol-disulfide exchange and unfolding are thermodynamically linked, the effects of a mutation (disulfide exchange) can be partitioned between various conformational states. In the case of unmodified V23C and the 2-hydroxyethyl protein mixed disulfide, significant effects in the nonnative states of nuclease are observed. Truncated forms of staphylococcal nuclease are thought to be partially folded and may be good models for early folding intermediates. We have characterized a truncated form of nuclease comprised of residues 1-135 with a V23C mutation after chemical modification of the cysteine residue. High-resolution size-exclusion chromatography indicates that modification brings about significant changes in the Stokes radius of the protein, and CD spectroscopy indicates considerable differences in the amount of secondary structure present. Measurement of the disulfide exchange equilibrium constant between this truncated protein and 2-hydroxyethyl disulfide indicate significant interactions between position 23 and the rest of the protein when the urea concentration is lower than 1.5 M.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immuno-capillary electrophoresis for the characterization of a monoclonal antibody against DNA

A monoclonal antibody against DNA established from a mouse strain that spontaneously develops systemic lupus erythematosus was characterized by migration shift immuno-capillary electrophoresis. The minimal size for DNA binding antibody was > 16 bases and the interaction with a double-stranded 32-mer oligonucleotide was almost one order of magnitude stronger than the interaction with a single-stranded oligonucleotide. The binding was highly dependent on the ionic strength conditions with an increase in binding with a decrease in ionic strength. The estimate of the dissociation constant for the antibody binding of a single stranded 32-mer oligonucleotide was 0.62 microM at pH 7.90. This value was in good agreement with the value of 0.44 microM measured by an independent method using biosensor (surface plasmon resonance) technology.