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R J Hamer

Publications and source records attributed to R J Hamer.

12 recordsLinked to original sources
PubMed2000

Formation of disulfide bonds in acid-induced gels of preheated whey protein isolate.

Cold gelation of whey proteins is a two-step process. First, protein aggregates are prepared by a heat treatment of a solution of native proteins in the absence of salt. Second, after cooling of the solution, gelation is induced by lowering the pH at ambient temperature. To demonstrate the additional formation of disulfide bonds during this second step, gelation of whey protein aggregates with and without a thiol-blocking treatment was studied. Modification of reactive thiols on the surface of the aggregates was carried out after the heat-treatment step. To exclude specific effects of the agent itself, different thiol-blocking agents were used. Dynamic light scattering and SDS-agarose gel electrophoresis were used to show that the size of the aggregates was not changed by this modification. The kinetics of gelation as determined by the development of pH and turbidity within the first 8 h of acidification were not affected by blocking thiol groups. During gelation, formation of large, covalently linked, aggregates occurred only in the case of unblocked WPI aggregates, which demonstrates that additional disulfide bonds were formed. Results of permeability and confocal scanning laser microscope measurements did not reveal any differences in the microstructure of networks prepared from treated or untreated whey protein aggregates. However, gel hardness was decreased 10-fold in gels prepared from blocked aggregates. Mixing different amounts of blocked and unblocked aggregates allowed gel hardness to be controlled. It is proposed that the initial microstructure of the gels is primarily determined by the acid-induced noncovalent interactions. The additional covalent disulfide bonds formed during gelation are involved in stabilizing the network and increase gel strength.

Acids↗
PubMed1998

Heat-induced conformational changes of patatin, the major potato tuber protein.

This paper presents a first structural characterization of isolated patatin, the major potato tuber protein, at ambient and elevated temperatures. Isolated patatin at room temperature is a highly structured molecule at both secondary and tertiary levels. It is estimated from far-ultraviolet circular dichroism data that about 33% of the residues adopts an alpha-helical and 46% a beta-stranded structure. Patatin is thermally destabilized at temperatures exceeding 28 degrees C, as was indicated by near-ultraviolet circular dichroism. It was shown that parts of the alpha-helical contributions unfold in the 45-55 degrees C region, whereas the beta-stranded parts unfold more gradually at temperatures of 50-90 degrees C. This was confirmed with Fourier-transform infrared spectroscopy. Differential scanning calorimetry indicated a cooperative transition between 50-60 degrees C, most likely reflecting the unfolding of alpha-helical parts of the molecule. Furthermore, fluorescence spectroscopy confirmed a global unfolding of the protein between 45-55 degrees C. The observed unfolding of the protein coincides with the inactivation of the patatin enzyme activity and with the precipitation as occurs in the potato fruit juice upon heating. At high temperatures, patatin still contains some helical and stranded structures. Upon cooling the protein partly refolds, it was observed that mainly alpha-helical structures were formed.

Butyrates↗
PubMed1997

Structure characterization of the central repetitive domain of high molecular weight gluten proteins. II. Characterization in solution and in the dry state.

The structure of the central repetitive domain of high molecular weight HMW) wheat gluten proteins was characterized in solution and in the dry state using HMW proteins Bx6 and Bx7 and a subcloned, bacterially expressed part of the repetitive domain of HMW Dx5. Model studies of the HMW consensus peptides PGQGQQ and GYYPTSPQQ formed the basis for the data analysis (van Dijk AA et al., 1997, Protein Sci 6:637-648). In solution, the repetitive domain contained a continuous nonoverlapping series of both type I and type II II beta-turns at positions predicted from the model studies; type II beta-turns occurred at QPGQ and QQGY sequences and type I beta-turns at YPTS and SPQQ. The subcloned part of the HMW Dx5 repetitive domain sometimes migrated as two bands on SDS-PAGE; we present evidence that this may be caused by a single amino acid insertion that disturbs the regular structure of beta-turns. The type I beta-turns are lost when the protein is dried on a solid surface, probably by conversion to type II beta-turns. The homogeneous type II beta-turn distribution is compatible with the formation of a beta-spiral structure, which provides the protein with elastic properties. The beta-turns and thus the beta-spiral are stabilized by hydrogen bonds within and between turns. Reformation of this hydrogen bonding network after, e.g., mechanical disruption may be important for the elastic properties of gluten proteins.

Amino Acid Sequence↗
PubMed1995

Shyness and public self-consciousness: additive or interactive relation with social interaction?

Shy people are characterized as engaging in self-derogatory thinking leading to anxiousness and inhibition, while people who are publicly self-conscious are focused on the impression they create on others. In addition, public self-consciousness has been described as an antecedent of shyness. In the present research, we tested additive versus interactive hypotheses about the association of shyness and public self-consciousness with dysfunctional social interaction. Undergraduate men varying in shyness and public self-consciousness engaged in a conversation with an unfamiliar woman confederate. Following the conversation, subjects completed self-report measures focusing on their responses during the conversation. Only main effects for shyness and public self-consciousness were found, supporting an additive hypothesis. Shyness was related to all dependent variables reflecting a negative self-bias, while public self-consciousness was not. In particular, shyness was inversely related to the balance of subjects' positive and negative thoughts and to reported use of protective as well as avoidance of acquisitive self-presentation responses. Public self-consciousness was positively related to use of protective self-presentation responses but unrelated to acquisitive responses.

Adult↗
PubMed1990

The effect of von Willebrand factor on activation of factor VIII by factor Xa.

Factor VIII has to be activated before it can serve efficiently as a cofactor in the intrinsic pathway of blood coagulation. This activation occurs through specific proteolytic cleavages in the molecule by either thrombin or factor Xa. In this study, we show that von Willebrand factor inhibits the activation of factor VIII by factor Xa. Incubation of factor VIII (30 U/ml) with 0.1 microgram/ml factor Xa resulted in a 1.6-fold activation followed by a decay of coagulant activity. In the presence of 10 micrograms/ml von Willebrand factor, activation and inactivation of factor VIII was completely inhibited. In contrast, the activation of factor VIII by thrombin was not influenced by von Willebrand factor. At high concentrations of factor Xa (10 micrograms/ml), von-Willebrand-factor-bound factor VIII could be cleaved and activated. The generated proteolytic fragments were identical to the fragments produced in the absence of von Willebrand factor and all fragments were released from von Willebrand factor. The major products were light-chain-derived fragments of molecular mass 66/68 kDa and 60 kDa and heavy-chain-derived fragments of 40 and 42 kDa. Also minor products of 12, 20/21, 23, 27 and 30 kDa were observed, most of which were specific for cleavage of factor VIII by factor Xa.

Factor VIII↗
PubMed1987

The effect of thrombin on the complex between factor VIII and von Willebrand factor.

Purified human factor FVIII (FVIII; 6000-8000 U/mg) was radiolabeled and bound to immobilized von Willebrand factor (vWF). The complex was incubated with human thrombin. Thrombin induced a release of 65% of the radioactivity initially bound. Released FVIII fragments and fragments remaining bound during incubation with thrombin were analyzed using gel electrophoresis. This led to the following observations. Released fragments largely consisted of Mr-70000 and Mr-50000 fragments; Mr-90000 and Mr-80000 fragments were only found in the fractions remaining bound to vWF and decreased with time. In contrast to these digestion products of FVIII, the Mr-42000 heavy-chain fragment remained bound to vWF, comprising the larger part of the radioactivity after a 2-h incubation. No thrombin-induced cleavages were observed in vWF. Furthermore, vWF-coated wells preincubated with thrombin were still able to bind 125I-FVIII. These results implicate a new concept for the activation of vWF-bound FVIII. Activation is a multistep process in which several cleavages are necessary to produce and release a coagulant-active FVIII molecule (FVIIIa), which is probably an Mr-50000/70000 heterodimer. Inactivation of FVIIIa is likely to be the result of a nonproteolytic dissociation due to loss of the joining divalent cation(s).

Electrophoresis, Polyacrylamide Gel↗
PubMed1987

Factor VIII binds to von Willebrand factor via its Mr-80,000 light chain.

The formation of a complex between factor VIII (FVIII) and von Willebrand factor (vWF) was studied using purified radiolabeled human FVIII and purified human vWF. A binding assay was developed in which vWF was coated on microtiter wells. FVIII was shown to bind specifically and reversibly to the immobilized vWF. At a coating of 70 pg vWF/well, binding was half-maximal at a FVIII concentration of 70 +/- 10 pM. In order to ascertain which part of FVIII interacted with vWF, eight monoclonal antibodies, directed against FVIII, were tested for their ability to inhibit FVIII-vWF interaction. One of the eight antibodies, CLB-CAg:58, inhibited binding completely. This antibody was demonstrated to react with the Mr-80,000 light chain of FVIII. Direct evidence for the involvement of this chain in vWF binding was obtained by studying the binding of isolated, radiolabeled FVIII heavy and light chains. In a typical experiment 23-30% of the radioactivity bound when the FVIII light chain was added and less than 1% when the FVIII heavy chain was added.

Antibodies, Monoclonal↗
PubMed1986

Human factor VIII: purification from commercial factor VIII concentrate, characterization, identification and radiolabeling.

Human factor VIII was purified from commercial factor VIII concentrate with a 12% yield. The specific coagulant activity of purified factor VIII was 8,000 units/mg. In the presence of SDS the purified factor VIII consisted of a variety of polypeptides on polyacrylamide gels, ranging between Mr 80,000 and Mr 208,000. In the absence of SDS the purified factor VIII showed an apparent molecular weight of 270,000 upon Sephadex G200 gel-filtration. The purified factor VIII could be activated by thrombin, which resulted in the disappearance of Mr 108,000-208,000 polypeptides in favor of an Mr 92,000 polypeptide. Treatment with factor Xa also activated factor VIII, whereas treatment with activated protein C resulted in the inactivation of coagulant activity. Coagulant-active 125I-factor VIII was prepared using a lactoperoxidase radioiodination procedure. This 125I-factor had the same characteristics as unlabeled factor VIII. All polypeptides could be precipitated with monoclonal antibodies directed against factor VIII. With 125I-factor VIII a pIapp of 5.7 was found in the presence of urea.

Chemical Precipitation↗
PubMed1986

The physiology and pathophysiology of the factor VIII complex.

The factor VIII complex consists of two noncovalently linked proteins: von Willebrand factor (VWF) and factor VIII (FVIII). VWF plays an important role in primary hemostasis by mediating the adherence of blood platelets to the damaged vessel wall. A review of the literature on VWF is given with regard to its physicochemical properties and mode of action. FVIII acts as a cofactor in the factor Xa-generating enzyme complex of the intrinsic coagulation cascade. Starting with the recently published primary structure of FVIII, the literature is reviewed for structural information on FVIII. Also, an effort is made to characterize the interaction of FVIII with VWF and to discuss the possible physiological significance of FVIII-VWF complex formation. Interaction of FVIII with the clotting factors of the intrinsic pathway of coagulation is described in detail. Hemophilia and von Willebrand's disease (VWD) are both congenital bleeding disorders affecting a great many people. The different variants of these diseases are described with some reference to therapy and detection.

Carbohydrates↗
PubMed1984

Functional domains on von Willebrand factor. Recognition of discrete tryptic fragments by monoclonal antibodies that inhibit interaction of von Willebrand factor with platelets and with collagen.

We have identified two functional domains on the von Willebrand factor (VWF) moiety of the Factor VIII-von Willebrand factor complex (FVIII-VWF), one interacting with blood platelets, and one interacting with vessel wall collagens, by means of two monoclonal antibodies directed against the VWF molecule, CLB-RAg 35 and CLB-RAg 201. The monoclonal antibody CLB-RAg 35 inhibited virtually all platelet adherence to artery subendothelium and to purified vessel wall collagen type III, at relatively high wall shear rates. CLB-RAg 35 also inhibited the ristocetin-induced platelet aggregation and the binding of FVIII-VWF to the platelet in the presence of ristocetin but did not affect the binding of FVIII-VWF to collagen. The monoclonal antibody CLB-RAg 201 inhibited the binding of FVIII-VWF to purified vessel wall collagen type I and III and all platelet adherence to collagen type III and the platelet adherence to subendothelium that was mediated by FVIII-VWF in plasma. The two functional domains on FVIII-VWF that were recognized by CLB-RAg 35 and CLB-RAg 201 were identified by means of immunoprecipitation studies of trypsin-digested FVIII-VWF. The domains resided on different polypeptide fragments, with a Mr of 48,000 for the collagen binding domain and a Mr of 116,000 for the platelet binding domain. The 116,000-mol wt fragment consisted of subunits of 52,000/56,000 mol wt and 14,000 mol wt after reduction. The 52,000/56,000-mol wt subunits possessed the epitope for CLB-RAg 35.

Antibodies, Monoclonal↗
PubMed1979

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Family Practice↗