Partial Heat Denaturation Research Articles

Correlation between CD16a binding and immuno effector functionality of an antigen specific immunoglobulin Fc fragment (Fcab)

Antigen binding immunoglobulin Fc fragments (Fcab) are generated by engineering loop regions in the CH3 domain of human IgG1 Fc. Variants of an Fcab specific for Her-2 were designed to display either enhanced (S239D:A330L:I332E) or diminished (L234A:L235A) binding affinities to the Fc receptor CD16a based on mutations described previously. The two mutant Fcab proteins demonstrated the expected modulation of CD16a binding. Interaction with recombinant or cell surface expressed Her-2 was unaffected in both mutants compared to the parental Fcab. Binding affinities for CD16a correlated with the ADCC-potencies of the Fcab variants. Additional studies indicated that the L234A:L235A variant Fcab had equivalent structural features as the unmodified Fcab since their DSC profiles were similar and antigen binding after re-folding upon partial heat denaturation had not changed. Introduction of the S239D:A330L:I332E mutations resulted in a significant reduction of the CH2 domain melting temperature, a moderate decrease of the thermal transition of the CH3 domain and lower antigen binding after thermal stress compared to the parental Fcab. We conclude that the known correlation between CD16a binding affinity and ADCC potency is also valid in Fcab proteins and that antigen specific Fcab molecules can be further engineered for fine tuning of immuno effector functions.

Mutations in CDON, Encoding a Hedgehog Receptor, Result in Holoprosencephaly and Defective Interactions with Other Hedgehog Receptors

Holoprosencephaly (HPE), a common human congenital anomaly defined by a failure to delineate the midline of the forebrain and/or midface, is associated with diminished Sonic hedgehog (SHH)-pathway activity in development of these structures. SHH signaling is regulated by a network of ligand-binding factors, including the primary receptor PTCH1 and the putative coreceptors, CDON (also called CDO), BOC, and GAS1. Although binding of SHH to these receptors promotes pathway activity, it is not known whether interactions between these receptors are important. We report here identification of missense CDON mutations in human HPE. These mutations diminish CDON's ability to support SHH-dependent gene expression in cell-based signaling assays. The mutations occur outside the SHH-binding domain of CDON, and the encoded variant CDON proteins do not display defects in binding to SHH. In contrast, wild-type CDON associates with PTCH1 and GAS1, but the variants do so inefficiently, in a manner that parallels their activity in cell-based assays. Our findings argue that CDON must associate with both ligand and other hedgehog-receptor components, particularly PTCH1, for signaling to occur and that disruption of the latter interactions is a mechanism of HPE.

Foam fractionation of α‐lactalbumin and β‐lactoglobulin from a whey solution

AbstractWhey, a byproduct of cheese production, is often considered a waste stream. Separation and purification of whey proteins is a difficult and expensive task. Occasionally, it is transported out of the dairy plant for a per volume charge. One possible method to reduce the waste volume and disposal cost is to concentrate whey by foam fractionation and potentially produce a valuable coproduct, a concentrated whey protein solution. Foam fractionation is an adsorptive bubble separation method based on the hydrophobic/hydrophilic properties of proteins. In this study, foam fractionation was evaluated for the concentration of whey proteins, specifically α‐lactalbumin and β‐lactoglobulin, from a dilute whey protein solution. The effects of initial whey protein concentration (0.075 and 0.15 mg/ml), pH (3.8–5.5), superficial gas velocity (0.85 and 0.95 cm/s) and temperature (4 and 65 °C) on protein enrichment and recovery were examined. Higher enrichment was achieved with the lower initial protein concentration (0.075 mg/ml), and at pH values that were near the isoelectric points (pI) of α‐lactalbumin and β‐lactoglobulin (pH 3.8, 4.2, 4.5, and 5.2). Higher superficial gas velocity enhanced the amount of proteins recovered with a decrease in the enrichment. Cold temperature treatment and partial heat denaturation of whey proteins reduced enrichment and increased protein recovery simultaneously. Copyright © 2009 Curtin University of Technology and John Wiley & Sons, Ltd.

BLM is required for faithful chromosome segregation and its localization defines a class of ultrafine anaphase bridges

Mutations in BLM cause Bloom's syndrome, a disorder associated with cancer predisposition and chromosomal instability. We investigated whether BLM plays a role in ensuring the faithful chromosome segregation in human cells. We show that BLM-defective cells display a higher frequency of anaphase bridges and lagging chromatin than do isogenic corrected derivatives that eptopically express the BLM protein. In normal cells undergoing mitosis, BLM protein localizes to anaphase bridges, where it colocalizes with its cellular partners, topoisomerase IIIalpha and hRMI1 (BLAP75). Using BLM staining as a marker, we have identified a class of ultrafine DNA bridges in anaphase that are surprisingly prevalent in the anaphase population of normal human cells. These so-called BLM-DNA bridges, which also stain for the PICH protein, frequently link centromeric loci, and are present at an elevated frequency in cells lacking BLM. On the basis of these results, we propose that sister-chromatid disjunction is often incomplete in human cells even after the onset of anaphase. We present a model for the action of BLM in ensuring complete sister chromatid decatenation in anaphase.

Temperature-mediated heteroduplex analysis for the detection of drug-resistant gene mutations in clinical isolates of Mycobacterium tuberculosis by denaturing HPLC, SURVEYOR nuclease

Denaturing high-performance liquid chromatography (DHPLC) is a relatively new technique, which utilizes heteroduplex formation between wild-type and mutated DNA strands to identify point mutations. Heteroduplex molecules are separated from homoduplex molecules by ion-pair, reverse-phase liquid chromatography on a special column matrix with partial heat denaturation of the DNA strands. In order to investigate the application of this method for point mutation detection in drug-resistant genes of Mycobacterium tuberculosis, katG, rpoB, embB, gyrA, pncA and rpsL genes, which are responsible for isoniazid, rifampicin, ethambutol, fluoroquinolone, pyrazinamide and streptomycin resistance, respectively, were detected by temperature-mediated DHPLC in 10 multidrug-resistant and 10 drug-susceptible clinical isolates. The DHPLC data were compared with those from a conventional MIC test. The results show that DHPLC is cost-effective with high capacity and accuracy, and is potentially useful for genotypic screening for mutations associated with anti-tuberculosis drug resistance.

Partial heat denaturation step during reverse transcription and PCR screening yields full-length 5'-cDNAs.

Partial heat denaturation step during reverse transcription and PCR screening yields full-length 5'-cDNAs.

Inactivation of gram-negative bacteria by lysozyme, denatured lysozyme, and lysozyme-derived peptides under high hydrostatic pressure.

We have studied the inactivation of six gram-negative bacteria (Escherichia coli, Pseudomonas fluorescens, Salmonella enterica serovar Typhimurium, Salmonella enteritidis, Shigella sonnei, and Shigella flexneri) by high hydrostatic pressure treatment in the presence of hen egg-white lysozyme, partially or completely denatured lysozyme, or a synthetic cationic peptide derived from either hen egg white or coliphage T4 lysozyme. None of these compounds had a bactericidal or bacteriostatic effect on any of the tested bacteria at atmospheric pressure. Under high pressure, all bacteria except both Salmonella species showed higher inactivation in the presence of 100 microg of lysozyme/ml than without this additive, indicating that pressure sensitized the bacteria to lysozyme. This extra inactivation by lysozyme was accompanied by the formation of spheroplasts. Complete knockout of the muramidase enzymatic activity of lysozyme by heat treatment fully eliminated its bactericidal effect under pressure, but partially denatured lysozyme was still active against some bacteria. Contrary to some recent reports, these results indicate that enzymatic activity is indispensable for the antimicrobial activity of lysozyme. However, partial heat denaturation extended the activity spectrum of lysozyme under pressure to serovar Typhimurium, suggesting enhanced uptake of partially denatured lysozyme through the serovar Typhimurium outer membrane. All test bacteria were sensitized by high pressure to a peptide corresponding to amino acid residues 96 to 116 of hen egg white, and all except E. coli and P. fluorescens were sensitized by high pressure to a peptide corresponding to amino acid residues 143 to 155 of T4 lysozyme. Since they are not enzymatically active, these peptides probably have a different mechanism of action than all lysozyme polypeptides.

Biologically Active Basic Fibroblast Growth Factor Migrates at 27 kD in “Non-Denaturing” SDS-Polyacrylamide Gel Electrophoresis

Using mild conditions of SDS-PAGE, i.e. no heating of the sample, and the PhastSystem (Pharmacia), we found that bFGF, either natural bovine or recombinant human migrated at a 27 kD position in addition to the classical 18 kD one. By the cell-blot technique, we found that the biological activity toward rat astroblasts and 3T3 mouse fibroblasts was always restricted to the 27 kD band. Partial heat denaturation experiments revealed a close correlation between the remaining biological activity of bFGF in solution and the ratio of the 27 kD band versus the 18 kD band seen on SDS gels. These observations suggest that the bFGF which is biologically active in solution migrates at an apparent Mr of 27 kD in our conditions of electrophoresis, keeping its biological activity after electrophoresis, and the molecules which are inactive (denatured) in solution migrate at 18 kD and remain inactive. These experimental conditions, in which the biological activity appears to be preserved, could be referred to as "non-denaturing SDS-polyacrylamide gel electrophoresis" and could be useful, associated to cell-blot, for the search and characterization of new growth factors active on cells in culture.

Both the 25-kDa and 50-kDa domains in myosin subfragment 1 are close to the reactive thiols.

The thiol-specific photoactivatable reagent benzophenone-4-iodoacetamide can be incorporated into myosin subfragment 1 (S1), accompanied by an increase of Ca2+-ATPase and the loss of K+-ATPase activities, a characteristic property of S1 when reactive sulfhydryl 1 (SH-1) is modified. After trypsin cleavage, 25-kDa, 50-kDa, and 20-kDa fragments were found upon NaDodSO4/polyacrylamide gel electrophoresis of the unphotolyzed sample, whereas only the 50-kDa fragment and a 45-kDa fragment appeared in the photolyzed sample, indicating that the NH2-terminal 25-kDa fragment was crosslinked to the COOH-terminal 20-kDa fragment via SH-1. When photolysis was carried out in the presence of Mg2+ and ATP or Mg2+ and adenosine 5-[beta, gamma-imido]triphosphate (AdoPP[NH]P), a 70-kDa band, attributable to a crosslinked (50 kDa + 20 kDa) species, was also observed. This suggests that the conformational change induced by nucleotide binding reduces the distance between the 50-kDa region and the label on SH-1. Similar results were obtained when labeling and photolysis were carried out on trypsin-nicked S1, in which the 25-kDa, 50-kDa, and 20-kDa fragments are held together noncovalently. Further, when labeling with benzophenone-4-iodoacetamide was carried out in the presence of Mg-ATP, which increases the reactivity of another thiol, presumably SH-2, both 45-kDa and 70-kDa species were formed upon photolysis in the absence of ATP, suggesting that SH-2 is close to the 50-kDa region. More of the 70-kDa species was formed, at the expense of the 45-kDa species, when photolysis was carried out in the presence of Mg-ATP. Partial heat denaturation preferentially reduced the crosslinking between the reactive thiols and the 50-kDa region.

Influence of temperature and ionic strength on the low-frequency dielectric dispersion of DNA solutions.

AbstractThe dielectric properties of DNA solutions at low frequencies (5 Hz to 2 kHz) have been measured by means of a four‐terminal bridge method utilized to minimize electrode polarization errors. At 24°C native salt‐free DNA has a very large specific dielectric increment, Δε/c = 9.8 × 106 l/mol and a very low frequency relaxation centered at 18 Hz. Both the dielectric increment and the relaxation time are greatly decreased by partial heat denaturation at temperatures above 60°C or by addition of salt, the effects being much larger for divalent anions. These results are shown to be in qualitative agreement with theoretical treatments of counterion fluctuation polarization by McTague and Gibbs for the equilibrium case and by Mandel for relaxation. The ratio of the relaxation time for the low‐frequency process to that previously observed at much higher frequencies suggests that these relaxations result from counterion fluctuations along the longitudinal and transverse axes of the molecule, respectively.

Infidelity of DNA synthesis by temperature-sensitive DNA polymerases from RNA tumor viruses

An analysis of the role of reverse transcriptase in base-selection has been carried out using mutant DNA polymerases from RNA tumor viruses. Three temperature-sensitive DNA polymerases from avian sarcoma viruses and a wild type DNA polymerase from an avian leukosis virus were assayed for the accuracy with which they copy homopolymers in vitro. With poly(A) · oligo(dT) as a template, the frequency of incorporation of the non-complementary nucleotide, dCMP, into the poly(dT) product varied from 1 in 235 to 1 in 276. With poly(C) · oligo(dG) as template, the incorporation of dAMP into poly(dG) varied from 1 in 875 to approx. 1 in 1700. Analyses of the polynucleotide products of the reaction by equilibrium density centrifugation and digestion with snake venom phophodiesterase indicate that the non-complementary nucleotides are incorporated in phosphodiester linkage and are distributed throughout the length of the product. With poly(A) and poly(C) templates, the rate of incorporation of both complementary and non-complementary nucleotides by each mutant DNA polymerase was temperature sensitive compared to that of wild type DNA polymerase. These data demonstrate that the polymerase, itself, catalyses the incorporation of both complementary and non-complementary nucleotides. Partial heat denaturation of the mutant polymerases did not change the fidelity of copying poly(A). With poly(C) as template, however, partial heat inactivation changed the error frequency of two of the mutant polymerases. These results suggest that the mutattions studied may alter base discrimination by the polymerase in a template-dependent manner.

Bovine enterokinase. Purification, specificity, and some molecular properties.

Enterokinase has been isolated from the contents of bovine duodena and purified 1200-fold in 41% yield. The isolation procedure employed DEAE chromatography, affinity chromatography on immobilized p-aminobenzamidine, and gel filtration. The resultant pure enzyme exhibits a single band on sodium dodecyl sulfate gel electrophoresis corresponding to a molecular weight of 145 000. Two chains in the molecule, connected by disulfide linkages, have molecular weights of 57 000 and 82 000, respectively. The purified enzyme exhibits a restricted specificity which is directed toward the polyanionic amino acid sequence in the activation peptide of the zymogen substrate. Of the zymogens of the serine proteases tested, including several of those involved in blood coagulation, only native and guanidinated trypsinogen are acitvated by enterokinase, whereas acetylated trypsinogen is not. Partial heat denaturation of bovine enterokinase causes a differential response toward the activation of trypsinogen and the hydrolysis of benzoyl-L-arginine ethyl ester (BZArgOEt), further suggesting that secondary sites are important in the binding of trypsinogen. A sensitive assay for enterokinase (nanomole range) was developed using tritiated BZArgOEt.

Electron microscopy study of length and partial denaturation of Rhizobium bacteriophage DNA.

By electron microscopy and melting of the DNA of some bacteriophages from the soil bacterium Rhizobium lupini, it was found that molecular weights range between 27 and 50 Mdaltons and GC contents between 53 and 62%. All DNAs studied are linear; one has exposed single-stranded terminals. Partial heat denaturation of two phage DNA permitted mapping of AT-rich sites within unique nucleotide sequences. The maps are asymmetric with respect to the midpoints of the DNA.

Mung Bean Nuclease I: V. A TOOL FOR PROBING LOW MELTING REGIONS IN DEOXYRIBONUCLEIC ACID

Abstract Partial heat denaturation of preparations of DNA of different origin produced regions that became susceptible to mung bean nuclease I. These regions were distributed throughout the whole molecule of DNA. The susceptible regions were richer in A and T than the over-all composition of the parent DNA, regardless of whether the parent was A + T-rich or C + G-rich DNA. In the digested material C = G, A = T, therefore both strands were attacked simultaneously.

Denaturation map of bacteriophage T7 DNA and direction of DNA transcription

After partial denaturation of bacteriophage T7 DNA by heat or alkali, electron microscopy showed preferential strand separation at A + T-rich sites which map asymmetrically at the following fractional lengths: 0.01, 0.16, 0.21, 0.26, 0.33, 0.42, 0.46, 0.57, 0.63, 0.66, 0.86, 0.91 and 0.99. This map physically distinguishes between the two molecular ends of T7 DNA. In vitro synthesis of RNA by Escherichia coli RNA-polymerase with T7 DNA as a template and subsequent partial heat denaturation of the complexed DNA permitted simultaneous localization of RNA-synthesis sites and of strand-separation sites on individual molecules. Since the enzyme is known to initiate close to the genetically defined left-end of T7 DNA, it was found that the fractional length 0 of the denaturation map is equivalent to the left-end of the genetic map and that at least 90% of RNA synthesis is initiated close to the left-end of T7 DNA.

The homogeneity of pig brain mitochondrial monoamine oxidase

The time-courses of inhibition of the activity of pig brain mitochondrial monoamine oxidase by 2-bromo-2-phenylacetaldehyde toward the substrates tyramine, tryptamine, 5-hydroxytryptamine, adrenaline and m- O-methylnoradrenaline have been found to be the same. Mixed substrate experiments gave K m values in agreement with those calculated by the usual methods, and the loss of activity resulting from partial heat denaturation of the enzyme was the same for all substrates. Harmine was shown to be a competitive inhibitor, and the K i was shown to be independent of the substrate used. These findings are interpreted as indicating that the majority of the monoamine oxidase activity in pig brain mitochondria is due to a single enzyme.

Studies on the specificity and mechanism of action of adenosine deaminase

The hydrolysis of various groups at the 6-position of derivatives of purine ribonucleoside, including adenosine, 2-amino- and N 6-methyladenosine, 6-chloro-, 6-methoxy-, 6-hydroxylamino-, 2-amino-6-chloro-, and 2-amino-6-methoxypuriue ribonucleoside, by adenosine deamiuase preparations from calf intestinal mucosa has been investigated, and K m and V m values have been determined. Evidence was produced that a single enzyme was involved in the hydrolysis of all these substrates. Thus, purine ribonucleoside inhibited the hydrolysis of each compound in a competitive manner, and K I values for this inhibitor in the presence of various substrates were in the narrow range of 0.75–1.16 × 10 −5 m. Moreover, partial heat denaturation of the enzyme decreased the activity toward each substrate to the same extent. Examination of the effect: of pH on k m suggested dissociating groups on the free enzyme with p K values of 5.3–6.1 and 9.5–10.2, and a dissociating group involving the enzyme substrate complex with a p K of 5.7–6.3. The enzyme was inactivated by p-hydroxymercuribenzoate. The involvement of an SH group at the active center was suggested since this inactivation was antagonized by the competitive inhibitor purine ribonucleoside. The great variation in V m values observed for the various substrates indicated that the rate limiting step was not the final hydrolysis of a common intermediate.