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C Frisch

Publications and source records attributed to C Frisch.

35 records · Page 2Linked to original sources
PubMed1997

Thermodynamics of the interaction of barnase and barstar: changes in free energy versus changes in enthalpy on mutation.

We have studied the thermodynamics of the interaction between the ribonuclease barnase and its natural polypeptide inhibitor barstar. The contribution of specific residues and interactions within the barnase-barstar interface to the enthalpy of binding has been examined using isothermal titration calorimetry and protein engineering. The enthalpy of association of the wild-type proteins is -18.9 (+/-0.1) kcal/mol at pH 8 and at 25 degrees C. The enthalpy of binding remains favourable for 31 different combinations of mutations in the interface. The effects on the binding enthalpy upon replacing a side-chain involved in the interaction of barnase and barstar are, however, always unfavourable and in most cases larger than the effects on the free energy of binding. Interaction enthalpies calculated by double mutant cycle analysis are in some cases much larger than the interaction free energies. The interaction enthalpies for complexes between different barnase mutants with amino acid substitutions of the general base residue glutamic acid 73 and a barstar variant (D39A) vary by as much as 8.3 kcal/mol while the coupling free energies differ only by 1 kcal/mol. The use of enthalpies for the analysis of structure-activity relationships appears to be complicated by enthalpy-entropy compensation of weak intermolecular interactions. These tend to cancel out in measurements of free energy, which is thus the preferred quantity for simple analysis of interactions.

Bacillus↗
PubMed1996

Contribution of the intramolecular disulfide bridge to the folding stability of REIv, the variable domain of a human immunoglobulin kappa light chain.

BACKGROUND: Immunoglobulin domains contain about 100 amino acid residues folded into two beta-sheets and stabilized in a sandwich by a conserved central disulfide bridge. Whether antibodies actually require disulfide bonds for stability has long been a matter of debate. The contribution made by the central disulfide bridge to the overall folding stability of the immunoglobulin REIv, the variable domain of a human kappa light chain, was investigated by introducing stabilizing amino acid replacements followed by removal of the disulfide bridge via chemical reduction or genetic substitution of the cysteine residues. RESULTS: Nine REIv variants were constructed by methods of protein engineering that have folding stabilities elevated relative wild-type REIv by (up to) 16.0 kJ mol-1. Eight of these variants can be cooperatively refolded after unfolding and chemical reduction of the disulfide bridge-in contrast to wildtype REIv. The stabilizing effect of one of these residue replacements (T39K) was rationalized by determining the structure of the respective REIv variant at 1.7 A. The loss of folding stability caused by reduction of the intramolecular disulfide bond is on average 19 kJ mol-1. Removal of the disulfide bridge by genetic substitution of C23 for valine resulted in a stable immunoglobulin domain in the context of the stabilizing Y32H amino acid exchange; again, REIv-C23V/Y32H has 18 kJ mol-1 less folding stability than REIv-Y32H. The data are consistent with the notion that all variants studied have the same overall three-dimensional structure with the disulfide bridge opened or closed. CONCLUSIONS: A comparison of the magnitude of the stabilizing effect exerted by the disulfide bond and the length of the mainchain loop framed by it suggests lowering of the entropy of the unfolded state as the sole source of the effect. Disulfide bonds are not necessary for proper folding of immunoglobulin variable domains and can be removed, provided the loss of folding stability is at least partly compensated by stabilizing amino acid exchanges.

Disulfides↗
PubMed1995

Immunoglobulin mutant library genetically screened for folding stability exploiting bacterial signal transduction.

A model repertoire of variants of immunoglobulin kappa variable domain REIv with different folding stabilities was generated by oligonucleotide-directed randomization of position 29, a key conserved residue of hypervariable loop 1. Fused to ToxR', the membrane-anchored cytoplasmic domain of the Vibrio cholerae ToxR transcription activator, different members of the library induce different levels of transcription from the ctx promoter in Escherichia coli. Differences in transcription activation correlate positively with folding stabilities of the corresponding REIv domains. Since conformationally stabilized REIv derivatives elicit a dark red colony phenotype on EMB-lactose indicator plates, this procedure constitutes a genetic screen for immunoglobulin folding stability.

Amino Acid Sequence↗
PubMed1995

Characterization of in vitro oxidized barstar.

The polypeptide inhibitor of the ribonuclease barnase, barstar, has two cysteine residues in positions 40 and 82. These have been proposed to form a disulfide bridge leading to an increase in stability without changing the inhibitory activity of the protein. Barstar and a mutant (E80A) were oxidized in vitro and the biochemical and physico-chemical properties of the oxidized monomers were analysed. The oxidized proteins show no inhibition of barnase using a plate assay and are significantly destabilized. CD spectra indicate a loss of secondary structure. The amino acid substitution E80 --> A stabilizes the oxidized barstar to about the same extent as it does the reduced protein, indicating, however, that the helical region which it is in is intact.

Bacterial Proteins↗
PubMed1995

Blockade of lithium chloride-induced conditioned place aversion as a test for antiemetic agents: comparison of metoclopramide with combined extracts of Zingiber officinale and Ginkgo biloba.

The present study tests the hypothesis that the blockade of lithium chloride-induced conditioned place aversion might be a suitable model to assess antiemetic properties of drugs, especially in species that do not vomit, like rats. The effects of the known antiemetic compound metoclopramide were compared with those of zingicomb, a combination preparation of extracts of Ginkgo biloba and Zingiber officinale, also presumed to have antiemetic properties. Place conditioning was performed using a conventional three-compartment test procedure. On three successive conditioning trials, rats received an intraperitoneal (i.p.) injection of lithium chloride (125 mg/kg) and were placed into the compartment that they had preferred over three baseline trials. During the test, rats treated with lithium chloride (LiCl) spent less time in the treatment compartment, indicative of a conditioned place aversion (CPA). In the first experiment, metoclopramide (MCP) was administered intragastrically (IG) in doses of 2 or 10 mg/kg 60 min prior to LiCl injection. The pretreatment with 50 and 100 mg/kg zingicomb attenuated the LiCl-produced CPA, whereas a dosage of 10 mg/kg had no effect. These findings suggest that LiCl-induced CPA is a viable procedure with which to assess the antiemetic properties of metoclopramide. Furthermore, the data confirm the hypothesis that the phytopharmacon zingicomb might have antiemetic properties that are comparable to those of metoclopramide.

Animals↗
PubMed1995

Facilitation of learning following injection of the chondroitin sulfate proteoglycan biglycan into the vicinity of the nucleus basalis magnocellularis.

The aim of this study was to examine the effects of biglycan, a small chondroitin sulfate proteoglycan with neurotrophic activity, on memory and reinforcement upon unilateral injection into the region of the nucleus basalis magnocellularis (NBM). In experiment 1, rats with chronically implanted cannulas were injected with biglycan and tested on the uphill avoidance task, which involves punishment of a high-probability turning response on a tilted platform (negative geotaxis). Immediately after the training trial, that is, after a tail-shock was administered upon performing the response, rats received one microinjection (0.5 microliter) of substance P (SP) in a reference dosage of 0.74 pmol or biglycan (doses ranging from 1.3 to 1300.0 nmol) into the NBM region. When tested 24 h later, rats treated with SP (0.74 pmol) or biglycan (2.1 and 2.6 nmol) had significantly longer uphill latencies than vehicle (PBS) controls, indicative of superior learning of the avoidance response. In experiment 2, a test for possible proactive effects of post-trial biglycan on performance during the retention trial was performed. Furthermore, the uphill avoidance task was combined with a conditioned place preference task to assess possible reinforcing effects of biglycan. Rats were injected with either 2.6 or 130.0 nmol biglycan immediately after the training trial of the uphill task. One control group received 2.6 nmol biglycan 5 h after the trial, a second group was sham-operated. Additional groups were included which received biglycan (2.6 or 130.0 nmol), SP (0.74 pmol) or PBS after the training trial but no tail-shock.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Self-administration of neurokinin substance P into the ventromedial caudate-putamen in rats.

There is evidence that the neurokinin substance P plays a role in learning and reinforcement processes. Reinforcing effects of substance P were found upon injection into several parts of the brain. The aim of the present study was to gauge possible reinforcing effects of microinjections of substance P into the ventromedial caudate-putamen in rats. Two different behavioral paradigms were employed. In the first experiment a two-compartment choice procedure was used and the rats could trigger substance P injections (500 pg per 5 nl injection volume) into the ventromedial caudate-putamen by entering one distinctive compartment. During the injection period, substance P-injected animals spent significantly more time in the drug-paired compartment than vehicle-injected controls. In the second experiment, nose-poking through a hole in one wall of the cage was used as the operant. Rats that could self-administer substance P (100 pg per 5 nl injection volume) into the ventromedial caudate-putamen emitted a significantly higher rate of operant responding on the first day of testing and a significantly lower rate on the third day compared to vehicle-injected animals. The experiments provide evidence that the administration of substance P into the ventromedial part of the caudate-putamen can have positive reinforcing effects, but that repeated injections can have aversive properties. These effects are discussed, firstly, with regard to the possible mechanisms of intrastriatal substance P on striatonigral and striatopallidal output systems and, secondly, with respect to their possible relevance in the study of the basal forebrain reinforcement system.

Animals↗
PubMed1994

Chronic administration of neurokinin SP improves maze performance in aged Rattus norvegicus.

Deficits in associative functions seen with senescence may be based, at least in part, on a decreased availability of trophic factors in the CNS. A reduced concentration of neurokinins, including undecapeptide substance P (SP), also accompanies aging. Thus, given the change in SP metabolism and the known mnemogenic as well as neurotrophic/neuroprotective effects of the peptide, it seems possible that age-related deficits in associative processes could be influenced by treatment with exogenous SP. In the present study, 30-month-old Wistar rats were injected daily with SP (50 or 250 micrograms/kg, intraperitoneally) starting 1 week before they were tested on the Morris water maze task and on motor coordination tests. Control groups included vehicle-injected old and adult (3-month-old) rats. Over the days of maze testing, application of the substances was performed 5 h after testing daily for 15 days and after the last drug delivery, maze testing was continued for 4 more days. The main finding of this study is that chronic administration of both dosages of SP (50 and 250 micrograms/kg) improved the maze performance of the old rats. This facilitatory effect of SP on performance was also evident after the drug treatment had been terminated in the course of maze testing. Furthermore, chronic application of SP in a dose range of 50-250 micrograms/kg was found to reduce age-related deficits in motor capacities.

Aging↗
PubMed1994

Dimerization of Bence Jones proteins: linking the rate of transcription from an Escherichia coli promoter to the association constant of REIV.

Homodimers of immunoglobulin VL domains are minimal models of antibodies in that they display an ensemble of six hypervariable loops. Bence Jones protein REI is a mixture of a complete kappa light chain and the corresponding variable domain (REIV). The known three-dimensional structure of the REIV dimer (Epp et al., 1975, Biochemistry 14, 4943-4952) provides a basis for studying dimer stabilization by protein engineering. Mutant REIV-L94H was constructed and shown to have an equilibrium constant of dimerization about one order of magnitude higher than wildtype REIV. By fusing REIV and variants to the aminoterminal part of the Vibrio cholerae ToxR regulator protein (Miller et al., 1987, Cell 48, 271-279), a transcriptional signal in E. coli can be derived from REIV homodimer formation constant. The system senses dimerization of the immunoglobulin part of the fusion protein, located in the periplasmatic space, and transduces the signal as transcriptional activation to a ctx::lacZ gene construct integrated into the E. coli chromosome. There is positive correlation between the propensities of homodimer formation and the rate of transcriptional initiation at the ctx promoter. Since beta-galactosidase levels can easily be measured colorimetrically in crude cell lysates of a large number of clones using an ELISA reader, this procedure constitutes all elements required for a genetic screen in E. coli for immunoglobulin variants with altered association constants.

Bacterial Proteins↗
PubMed1994

A soluble immunoglobulin variable domain without a disulfide bridge: construction, accumulation in the cytoplasm of E. coli, purification and physicochemical characterization.

Two amino acid exchanges (Y32H and C23V) were introduced sequentially into the immunoglobulin REIV, a human kappa variable domain. The first exchange stabilizes the folded state of the domain by 4.6 kJ/mol (1.1 kcal/mol), the second abolishes the central disulfide bridge and destabilizes the folded domain by 17.5 kJ/mol (4.2 kcal/mol). Introduction of the stabilizing exchange first is a necessary pre-requisite to the removal of the central disulfide bridge without collapse of the fold. The double mutant REIV-C23V/Y32H can be accumulated in the cytoplasmatic compartment of the E. coli cell, a finding that opens new possibilities in antibody engineering.

Base Sequence↗
PubMed1993

Studies of the protein synthesis system in the brain cortex during global ischemia and reperfusion.

Previous studies have demonstrated that brain protein synthesis declines after global ischemia and reperfusion. To investigate the role of the translation system in this phenomenon, we examined the ability of partially purified ribosomes, ribosome-bound mRNA and translation cofactors derived from the transiently ischemic cerebral cortex to synthesize protein in vitro. Samples were prepared from canines subjected to 20-min cardiac arrest and after 2 or 8 h of post-resuscitation intensive care. There was no significant decrease in the rate of in vitro protein synthesis as a consequence of either ischemia or reperfusion. Northern hybridization of ribosome-bound RNA revealed a discrete band of mRNA for brain-specific creatine kinase (ck-bb) that was consistent in presence and intensity in all groups. However, mRNA for heat shock 70 protein (hsp-70) was observed only during reperfusion and markedly increased between 2 and 8 h reperfusion. Thus, we conclude that (1) the transcription system is intact during reperfusion and hsp-70 mRNA is made and translocated to the ribosomes during reperfusion, (2) mRNA for ck-bb is not displaced from ribosomes by the appearance of hsp-70 during reperfusion and (3) isolated ribosomes maintain their ability to translate in vitro during the first 8 h of reperfusion after global brain ischemia. Therefore, the early reduction in protein synthesis observed in vivo during post-ischemic brain reperfusion is not due to an intrinsic dysfunction of the ribosomes.

Animals↗
PubMed1993

[Sonography of the salivary glands].

358 sonographic studies of the salivary glands of 255 patients with proven diagnoses were evaluated retrospectively. Sonography proves to be highly valuable to differentiate between peri- and intraglandular lesions (98%). Superficial neoplasms can be delineated easily. A sharp margin of the tumour is not reliable to predict benignity, therefore biopsy should be performed in all neoplasms of the glands. Deep infiltrating processes require further evaluation by CT or MR. In cases with sialolithiasis the stones can be demonstrated in approx. 2/3 by ultrasound. Inflammatory diseases of the salivary glands have variant sonographic features. Acute infections usually have hypoechogenic parenchyma, chronic inflammations are more likely hyperechogenic.

Adenolymphoma↗
PubMed1990

Occupational exposure to amorphous silica dust and pulmonary function.

Respiratory manifestations among 41 workers exposed to amorphous silica dust were compared with a control group comprising 90 workers of equivalent socioeconomic state in the same plant. Flow volumes were determined, blood gas concentrations were measured at rest and during exercise, chest radiographs were obtained, and data about respiratory symptoms were collected by questionnaire. A dust exposure index was calculated for each exposed worker. It was not possible to differentiate between the two groups from the questionnaire, blood gas analysis, or chest radiographs. On the other hand, the tests of respiratory function showed a significant decrease in forced expiratory flow (FEF25-75, FEF50, and FEF75) in the exposed group compared with the controls, although no correlation was found between the exposure index and pulmonary function. It appears that smoking and exposure to amorphous silica synergise to induce small airway disease.

Adult↗
PubMed1984

Improved results in acute appendicitis care following areawide review.

The Wisconsin Professional Review Organization compared acute appendectomies being performed in 1981 to those done in 1978 in 32 Wisconsin hospitals. In both years approximately 75 percent of primary appendectomies were in patients 5 to 30 years of age, one-fourth were in patients 15 to 19 years of age, and the majority were in males. Incidence of normal appendices dropped from 16.1 percent in 1978 to 11.4 percent in 1981 (p less than 0.005). The number of patients with normal appendices who did not meet symptom criteria dropped from 37.3 percent to 9.5 percent (p less than 0.05). Incidence of normal appendices was highest in small hospitals. Severity and ruptures or perforations increased, but not significantly. Postoperative complications and mortality decreased. Average length of stay decreased overall, but increased for patients with complications and ruptures or perforations. These data suggest that areawide reviews assure quality and help contain costs. Physician self-regulation using areawide studies may produce desirable change.

Acute Disease↗
PubMedDate not supplied

[Inhibition of degranulation of human basophils by calcium antagonists].

The human basophil degranulation test (HBDT) quantifies the apparent disappearance of basophils after contact with the sensitizing allergen. The inhibition of degranulation by pharmacological products can be appreciated by incubating beforehand the basophils with the substance concerned during various times. The inhibitory effect is evaluated by the difference of degranulation with or without it. EDTA, sodium cromoglycate, verapamil and bepridil have been tested at different concentrations and at 3 times of basophils preincubation (0,15 and 30 minutes). Results obtained confirm previous reports concerning cromoglycate which didn't inhibit degranulation of basophils. EDTA at 3.4*10(-2)M and 3.4*10(-3)M concentration has an inhibitory effect, not increasing with the time of preincubation. The inhibition with verapamil is of the same order at 1*10(-4)M for the 3 times of incubation and weaker, but significant, at 1*10-M5 after 30 minutes of preincubation. Bepridil inhibits at 1*10(-4)M, but this effect disappears if basophils are preincubated 30 minutes with the drug. The inhibition of the specific basophil degranulation by these drugs is probably du to their calcium antagonist property. However their effect is not quite similar when cells are preincubated at different times with the drug. On the other hand, one can study with HBDT the inhibitory effect of a new product, even if the therapeutic indications would have to be studied afterwards.

Basophils↗