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M Beauregard

Publications and source records attributed to M Beauregard.

At least 37 records · Page 2Linked to original sources
PubMed1999

Characterization of MB-1. A dimeric helical protein with a compact core.

MB-1 is a de-novo protein designed to incorporate a large number of the nutritionally important amino acids methionine, lysine, leucine and threonine into a stable four-helix bundle protein. MB-1 has been expressed and purified from Escherichia coli, indicating it was resistant to intracellular proteases [Beauregard, M., Dupont, C., Teather, R.M. & Hefford, M.A. (1995) Bio/Technology 13, 974]. Here we report an analysis of the secondary, tertiary and quaternary structures in MB-1 using circular dichroism, fluorospectroscopy and size-exclusion chromatography. Our data indicate that the MB-1 structure is close to the target structure, an alpha-helical bundle, in many respects and is highly helical in solution. The single tyrosine incorporated into the designed protein as a spectrocopic probe of tertiary structure, is buried in a compact, folded core and becomes accessible on protein denaturation, as per design. Furthermore, MB-1 was found to be native-like in many respects: (a) protein denaturation induced by urea is cooperative and fully reversible; (b) its oligomeric state at moderate concentration is well defined; and (c) MB-1 has very low affinity for 8-anilino-1-naphthalenesulfonic acid (ANSA), leading to enhancement of ANSA fluorescence that resembles that of other native proteins. On the other hand, our analysis revealed two aspects that command further attention. The folding stability of MB-1 as assessed by urea and thermal denaturation is somewhat less than that found for natural globular proteins of similar size. Size-exclusion chromatography experiments and analysis of MB-1 denaturation indicate that MB-1 is dimeric, not monomeric as designed. In light of these results, the utility and the current limitations of our design approach are discussed.

Chromatography, Gel↗
PubMed1999

The neural substrate of picture naming.

A PET study of 10 normal males was carried out using the bolus H215O intravenous injection technique to examine the effects of picture naming and semantic judgment on blood flow. In a series of conditions, subjects (1) passively viewed flashing plus signs, (2) noted the occurrence of abstract patterns, (3) named animal pictures, or (4) carried out a semantic judgment on animal pictures. Anticipatory scans were carried out after the subjects were presented with the instructions but before they began the cognitive task, as they were passively viewing plus signs. Our results serve to clarify a number of current controversies regarding the neural substrate of picture naming. The results indicate that the fusiform gyrus is unlikely to be the region where low-level perceptual processing such as shape analysis is undertaken. In fact, our evidence suggests that activation of the fusiform gyrus is most likely related to visual perceptual semantic processing. In addition, the inferior/middle frontal lobe activity observed while performing the picture naming and semantic judgment tasks does not appear to be due to the effects of anticipation or preparation. Furthermore, there appears to be a set of regions (a semantic network) that becomes activated regardless of whether the subjects perform a picture naming or semantic judgment task. Finally, picture naming of animals did not activate either parietal regions or anterior inferior left temporal regions, regardless of what subtraction baseline was used.

Adult↗
PubMed1998

The functional neuroanatomy of major depression: an fMRI study using an emotional activation paradigm.

An important issue regarding the neural basis of major depression is whether the functional brain changes associated with the affect disturbance seen in this syndrome are similar to those that accompany transient sadness in normal subjects. To address this question, we carried out an fMRI study using an emotional activation paradigm. Brain activity associated with passive viewing of an emotionally laden film clip aimed at inducing a transient state of sadness was contrasted with that associated with passive viewing of an emotionally neutral film clip in patients suffering from unipolar depression and in normal control subjects. Results showed that transient sadness produced significant activation in the medial and inferior prefrontal cortices, the middle temporal cortex, the cerebellum and the caudate in both depressed and normal subjects. They also revealed that passive viewing of the emotionally laden film clip produced a significantly greater activation in the left medial prefrontal cortex and in the right cingulate gyrus in depressed patients than in normal control subjects. These findings suggest that these two cortical regions might be part of a neural network implicated in the pathophysiology of major depression. Taken together, these results strongly support the view that activation paradigms represent an extremely useful and powerful way of delineating the functional anatomy of the various symptoms that characterize major depression.

Adult↗
PubMed1998

Crystallization and stabilization of MB-1, a de novo designed protein for optimized feeding technology.

Milk Bundle-1 is a de novo protein that was designed for application in agriculture. It has a high content of selected essential amino acids, and is intended to adopt an alpha-helical bundle fold. Crystallization experiments with MB-1 have been carried out on the ground and in reduced gravity on board Columbia orbiter during mission STS-80. Rather small crystals were obtained (< 0.05 mm) in both environments. Among other factors, the lack of stability of purified MB-1 has been detrimental to crystal growth. We report here on our progress with regard to optimizing crystal growth conditions, protein purification and protein stability. The first MB-1 mutant we present (MB-1-His) contains a poly-histidine tail, allowing the use of metal affinity chromatography for purification. MB-1-His has been found to keep its original mass for a month at room temperature, a spectacular improvement over MB-1. The other mutant (MB-1-Cys) was engineered to carry a cysteine residue on a solvent exposed face. The exposed cysteine binds readily to p-HMB, and allows for dimerization of MB-1-Cys. The dimer was found to be twice as stable as MB-1 during proteolytic degradation studies.

Amino Acid Sequence↗
PubMed1998

A role for the hippocampal formation in implicit memory: a 3-D PET study.

The apparent preservation of word priming effects in amnesia has been interpreted as supporting the view that implicit memory depends on brain systems that are independent of mesial temporal lobe structures which are in part responsible for explicit memory disorders. Nevertheless, a number of studies have demonstrated word priming deficits in amnesic patients relatively to normal subjects, suggesting that such structures may also be involved in implicit memory. To determine whether one such structure, the hippocampal formation, is a component of the brain system subserving word priming, a 3-D PET study was carried out in 13 normal individuals. Encoding was carried out using the brief multiple presentation technique, a procedure that allows one to effectively circumvent contamination of implicit memory tasks by explicit memory strategies. Results revealed that word priming was indeed associated with an activation of the right hippocampal formation. This finding of an hippocampal involvement in word priming calls into question the notion of absolute dissociability between the brain systems underlying performance on various explicit and implicit memory tasks.

Adolescent↗
PubMed1998

Addition of a poly-(6X) His tag to Milk Bundle-1 and purification using immobilized metal-affinity chromatography.

Milk Bundle 1 (MB-1) is a de novo designed protein enriched in M, T, K, and L. Its future application is as a high-quality dietary protein source for ruminants. The protein is currently expressed in Escherichia coli and is being characterized to solve its folded conformation. MB-1 has marginal stability at room temperature, which has hindered our attempts at characterization. To increase the stability of the protein at room temperature, the purification procedure was examined and changed to hopefully increase its effectiveness. We describe here the production and purification of a new MB-1 with six His residues at the C-terminal end. This allows the new mutant (MB-1-His) to bind metal ions and to be purified with immobilized metal-affinity chromatography (IMAC). MB-1-His obtained using IMAC was purer on SDS-PAGE than both MB-1 or MB-1-His isolated using the current protocol. The IMAC protocol is more economical and more efficient; preliminary results show that the protein purified by this method is also quite stable at room temperature.

Amino Acid Sequence↗
PubMed1998

Fluorescence spectroscopy monitoring of the conformational restraint of formaldehyde- and glutaraldehyde-treated infectious bursal disease virus proteins.

Interaction of native proteinaceous antigens during the recognition and the effector phases of an immune response leads to antigenic conformational modifications which may elicit additional specific immune response. Protein cross-linking and conformation restraining formaldehyde and glutaraldehyde have been extensively used in vaccine preparation, but the relative efficiencies of conformational restraint at concentrations similar to those used in vaccine preparation have not been investigated. We addressed this issue by comparing the extent of conformational restraint of virus proteins in formaldehyde- and glutaraldehyde-treated virus preparations by monitoring the fluorescence intensities (I320) of infectious bursal disease virus preparations (IBDV) and those of untreated virus during thermal denaturation. Formaldehyde was found to cause no detectable conformational restraint at 0.01% and only very weak restraint at 1%, while glutaraldehyde caused very strong conformational restraint at 0.01%. It is proposed how conformational restraint of proteinaceous antigens may alter ensuing immunity.

Animals↗
PubMed1997

Prediction of folding stability and degradability of the de novo designed protein MB-1 in cow rumen.

The authors have recently reported on the design of a protein (MB-1) enriched in methionine, threonine, lysine, and leucine. The protein is intended to be produced by rumen bacteria, in a way that would provide high producing lactating cows with limiting amino acids. In this report, MB-1 stability in the rumen is assessed, i.e., where the protein might be found after cell lysis or after being secreted by rumen bacteria. Current in vitro methods used to predict proteolytic degradability in the rumen were used for MB-1, as well as other natural proteins for comparison. MB-1 was found to be more susceptible to degradation than cytochrome c and ribonuclease A. Data indicate that MB-1 will be rapidly degraded if exposed to the rumen environment without protection. The contribution of folding stability to proteolytic stability was also examined. Rumen liquor components were selected to formulate a solution compatible with constraints of thermal denaturation studies. Denaturation curves show that the natural proteins were folded at rumen temperature. The MB-1 denaturation curves indicated that MB-1 does not unfold in a cooperative transition when heated from 20 to 70 degrees C. This suggests that MB-1 structure may be progressively modified as temperature increases, and that a continuum of conformations are available to MB-1. At 39 degrees C, a significant (50%) portion of MB-1 molecules had their tertiary structure unfolded, contributing to proteolytic degradability. Despite the unusual constraints used in MB-1 design (i.e., a maximized content in selected essential amino acids), results show that MB-1 has structural properties similar to previously reported de novo designed proteins.

Amino Acids, Essential↗
PubMed1997

Word priming with brief multiple presentation technique: preservation in amnesia.

Many studies have shown relative preservation of word priming in subjects with mild amnesia, but some decrease in severe amnesia. This calls into question the degree of separation between implicit and explicit memory. Possible contamination of implicit memory tasks by impaired explicit memory strategies might be obscuring the actual dissociation between the two memory systems. We have developed a method of circumventing explicit memory contamination by using brief duration repeated primes below the awareness threshold of subjects. We have used this approach to evaluate the status of word priming in densely amnesic subjects. One group of amnesic subjects with alcoholic Korsakoff's syndrome and one group of normal elderly control subjects were tested for word priming on a speeded category membership decision task. Implicit or explicit encoding procedures were used in three different experiments. Results demonstrated that brief multiple presentation of words can offer a means of producing word priming in the absence of explicit recognition or recall of the primed words in both amnesic subjects and normal elderly control subjects. Moreover, there was no significant difference in the magnitude of the priming effect between these groups in the three experiments. These findings show that amnesic subjects can exhibit normal levels of word priming. They also suggest that amnesics retain the capacity to encode, store and retrieve information implicity, e.g. unintentionally.

Aged↗
PubMed1997

Detection of an unfolding intermediate in alpha-urease with enhanced affinity for ANSA.

Protein aggregation is believed to be due to conformers that expose hydrophobic clusters that promote protein association. Such conformers can be detected using a fluorescent probe like 8-anilino 1-naphthalenesulfonic acid (ANSA). Here we show that urease exposed to 1.0 M guanidine-hydrogen chloride has a higher affinity for ANSA that native or denatured urease. The binding occurs over a narrow range of denaturant concentration, well below the concentration required to induce denaturation. The impact of ANSA on urease aggregation was further studied by fluorescence, light-scattering, and activity measurements. We found that ANSA modifies urease aggregates and can provide partial protection against inactivation arising from thermally induced aggregation. It seems that the well-known susceptibility of urease to aggregation is due to an intermediate that can be populated in the absence of denaturation. Such a rationale would explain why folding stability of urease is a poor indicator of long-term stabilization by various media.

Anilino Naphthalenesulfonates↗
PubMed1996

Three hTIM mutants that provide new insights on why TIM is a dimer.

Human triosephosphate isomerase (hTIM), a dimeric enzyme, was altered by site-directed mutagenesis in order to determine whether it can be dissociated into monomers. Two hTIM mutants were produced, in which a glutamine residue was substituted for either Met14 or Arg98, both of which are interface residuces. These substitutions strongly interfere with TIM subunit association, since these mutant TIMs appear to exist as compact monomers in dynamic equilibrium with dimers. In kinetic studies, the M14Q mutant exhibits significant catalytic activity, while the R98Q enzyme is inactive. The M14Q enzyme is nevertheless much less active than unmutated hTIM. Moreover, its specific activity is concentration dependent, suggesting a dissociation process in which the monomers are inactive. In order to determine the conformational stability of the wild-type and mutant hTIMs, unfolding of all three enzymes was monitored by circular dichroism and tryptophan fluorescence spectroscopy. In each case, protein stability is concentration dependent, and the unfolding reaction is compatible with a two-state model involving the native dimer and unfolded monomers. The conformational stability of hTIM, as estimated according to this model, is 19.3 (+/-0.4) kcal/mol. The M14Q and R98Q replacements significantly reduce enzyme stability, since the free energies of unfolding are 13.8 and 13.5 (+/- 0.3) kcal/mol respectively, for the mutants, A third mutant, in which the M14Q and R98Q replacements are cumulated, behaves like a monomer. The stability of this mutant is not concentration-dependent, and the unfolding reaction is assigned to a transition from a folded monomer to an unfolded monomer. The conformational stability of this double mutant is estimated 2.5 (+/-0.1) kcal/mol. All these data combined suggest that TIM monomers are thermodynamically unstable. This might explain why TIM occurs only as a dimer.

Amino Acid Sequence↗
PubMed1996

Neonatal insult to the hippocampal region and schizophrenia: a review and a putative animal model.

OBJECTIVE: To review the mounting evidence implicating early hippocampal dysfunction in the pathogenesis and the pathophysiology of schizophrenia. An account is made of recent neurodevelopmental hypotheses indicating how an early dysfunction of the hippocampal region disrupts maturational events in brain systems connected to that structure, thus inducing dysfunctional connectional development. Finally, an animal model is presented. METHOD: Socioemotional behaviour of monkeys (Macaca mulatta) with selective neonatal hippocampal lesions was assessed by analyzing their interactions with their age-matched controls at 2 months, 6 months, and 5 to 8 years of age and by comparing the social interactions at each age with those of normal controls paired together. RESULTS: At 2 months of age, monkeys with neonatal hippocampal lesions presented minor disturbances in initiation of social interactions. These subtle changes of behaviour were less evident at 6 months, although by that age, the operated monkeys displayed more withdrawals in response to an increase in aggressive responses from their unoperated peers. In adulthood, the amount of time spent by the hippocampectomized monkeys in social contacts with their normal peers decreased markedly. In addition, operated monkeys exhibited more locomotor stereotypies than normal controls. CONCLUSION: These experimental findings indicate that the time-course and nature of the behavioural disturbances resulting from early trauma to the hippocampal region have some similarities with the clinical symptoms of schizophrenic patients and the typical time-course of the disease.

Animals↗
PubMed1995

Stereotypies and loss of social affiliation after early hippocampectomy in primates.

The present study was aimed at determining whether early hippocampal damage alters the development of normal social interactions. Results showed that, at 2 months of age, animals with neonatal hippocampal lesions presented minor disturbances in initiation of social interactions. These subtle changes in behavior were less evident at 6 months, although at this age, the operated animals displayed more withdrawals in response to an increase in aggressive responses from their unoperated peers. Finally, in adulthood, the amount of time spent by the operated monkeys in social contacts with their normal peers was markedly less than that in normal dyads. Only in adulthood did the operated animals exhibit more locomotor stereotypies than normal controls. This finding suggest that the hippocampal formation may directly or indirectly affect the maintenance of social bounds in primates.

Animals↗
PubMed1995

Design, expression, and initial characterization of MB1, a de novo protein enriched in essential amino acids.

Using recently emerging protein folding principles we have designed a protein enriched in the essential amino acids methionine, threonine, lysine and leucine. Our preliminary study of consensus residues (based on charge, hydrophobicity and volume) of natural alpha-helical bundle proteins indicated that the residues M, T, K, and L could be inserted in an alpha-helical bundle structure. We therefore attempted to create a stable de novo protein, highly enriched in these essential amino acids, that would adopt the alpha-helical bundle fold. The design process was an iterative one. The consensus residues (based on the properties profile) for bundle helices were found considering the four helices taken together, helices I to IV individually, or only their N- and C-termini. Using these data, the helices in our de novo protein were designed by inserting the residues M, T, K and L as often as possible at positions where their volume, hydrophobicity and charge match the consensus found in natural bundle helices. Short sequences of strong turn formers were used to join the helices and adjust the predicted p1 to 7.7, while a number of local and global factors were used to refine our design. Further, the sequence was checked to eliminate various known protease targets in E. coli. The sequence of our de novo protein, MB1, is: MAT-EDMTDMMTTLFKTMQLLTK-SEPTA-MDEATKTATTMKNHLQNLMQK-TKNKE DMTDMATTYFKTMQLLTK-TEPSA-MDEATKTATTMKNHLQNLMQK-GVA+ ++ , where dashes separate long helices from short, turn forming linkers. A gene coding for this protein was assembled from synthetic oligonucleotides, then fused to the maltose binding protein gene under the control of a tac promoter. The fusion protein was expressed in E. coli, purified and cleaved to yield maltose binding protein and our de novo protein, MB1. MB1 was found to be helical, to have the expected molecular weight (11 kDa) and the expected content (57%) of the essential amino acids M, T, K and L.

ATP-Binding Cassette Transporters↗
PubMed1994

Detection of alpha-urease dissociation by fluorescence emission spectroscopy.

alpha-Urease (E.C. 3.5.1.5) is a hexameric enzyme that can dissociate into two active trimers and further into six monomers (Hirai M., Kawa-Hirai, R., Hirai T., Ueki T. (1993) Eur. J. Biochem. 215, 55-61). We have attempted to detect these dissociation products using fluorescence emission and polarization spectroscopy. Our data suggest that the hexamer, the trimer and denatured monomer have fluorescence emission maximum at 330nm, 332nm, and 346nm, respectively, under our experimental conditions. We have also found that the conformation of urease subunits is only slightly modified upon dissociation to trimer, but drastically changed upon further dissociation leading to monomer.

Fluorescence Polarization↗