Low crowding agent concentration destabilizes against pressure unfolding.

The concentration of macromolecules inside a cell is very high, which can affect the behavior of the enzymes, and consequently influence vital biological processes. This is called macromolecular crowding. Since the most important effect of macromolecular crowding is the excluded volume, we performed pressure experiments, where the volume (as conjugate parameter to the pressure) is the crucial factor. We measured the temperature and pressure stability of bovine serum albumin and lysozyme with various concentrations of crowding agents, dextran, Ficoll™ and lysozyme itself. Our most interesting finding is that low concentration of all the studied crowding agents decreases the pressure stability of the proteins. We explain this by the reduced hydration volume change in the crowded environment. Furthermore, we discuss the volumetric parameters and emphasize the difference between the partial volume of the protein and the volume it influences, and their relation to the excluded volume which is responsible for the macromolecular crowding.

The chaperonins of Synechocystis PCC 6803 differ in heat inducibility and chaperone activity.

The chaperonins GroEL and Cpn60 were isolated from the cyanobacterium Synechocystis PCC 6803 and characterized. In cells grown under optimal conditions their ratio was about one to one. However, the amount of GroEL increased considerably more than that of Cpn60 in response to heat stress. The labile chaperonin oligomer required stabilization by MgATP or glycerol during isolation. Use of the E. coli mutant strain, groEL44 revealed that the functional properties of the two cyanobacterial chaperonins are strikingly different. Overexpression of cyanobacterial GroEL in the E. coli mutant strain allowed growth at elevated temperature, the formation of mature bacteriophage T4, and active Rubisco enzyme assembly. In contrast, Cpn60 partially complemented the temperature-sensitive phenotype, the Rubisco assembly defect and did not promote the growth of the bacteriophage T4. The difference in chaperone activity of the two cyanobacterial chaperonins very probably reflects the unique chaperonin properties required during the life of Synechocystis PCC 6803.

Synechocystis HSP17 is an amphitropic protein that stabilizes heat-stressed membranes and binds denatured proteins for subsequent chaperone-mediated refolding.

The small heat shock proteins (sHSPs) are ubiquitous stress proteins proposed to act as molecular chaperones to prevent irreversible protein denaturation. We characterized the chaperone activity of Synechocystis HSP17 and found that it has not only protein-protective activity, but also a previously unrecognized ability to stabilize lipid membranes. Like other sHSPs, recombinant Synechocystis HSP17 formed stable complexes with denatured malate dehydrogenase and served as a reservoir for the unfolded substrate, transferring it to the DnaK/DnaJ/GrpE and GroEL/ES chaperone network for subsequent refolding. Large unilamellar vesicles made of synthetic and cyanobacterial lipids were found to modulate this refolding process. Investigation of HSP17-lipid interactions revealed a preference for the liquid crystalline phase and resulted in an elevated physical order in model lipid membranes. Direct evidence for the participation of HSP17 in the control of thylakoid membrane physical state in vivo was gained by examining an hsp17(-) deletion mutant compared with the isogenic wild-type hsp17(+) revertant Synechocystis cells. We suggest that, together with GroEL, HSP17 behaves as an amphitropic protein and plays a dual role. Depending on its membrane or cytosolic location, it may function as a "membrane stabilizing factor" as well as a member of a multichaperone protein-folding network. Membrane association of sHSPs could antagonize the heat-induced hyperfluidization of specific membrane domains and thereby serve to preserve structural and functional integrity of biomembranes.

[The importance of gender in the gynecologist-patient relationship]. / A nem jelentösége nögyógyász és betege kapcsolatában.

One of the focal interest of medical psychology is the quality of doctor-patient relationship. The sex of physician and patient have an important impact on their interaction. From this point of view, the contact of gynaecologist and client has a proper character. The authors review the references of this topic. They list the differences between male and female physicians, they also list the factors which determine the patient's choice of one or an other gynaecologist, finally authors deal with the motivations to become a gynaecologist. Both the male and female gynaecologist apply psychological egodefence mechanisms to elaborate sexual-erotic impulses. The adequate relationship needs a convergent, complementer interaction between doctor and patient. According to observations, patients are more satisfied by the female gynaecologists. Obstetricians have to give up the traditional and rigid view of gender and doctor's role. Balint-groups could help to develop and maintain more adequate doctor-patient relationship.

Membrane physical state controls the signaling mechanism of the heat shock response in Synechocystis PCC 6803: identification of hsp17 as a "fluidity gene".

The fluidity of Synechocystis membranes was adjusted in vivo by temperature acclimation, addition of fluidizer agent benzyl alcohol, or catalytic lipid hydrogenation specific to plasma membranes. The reduced membrane physical order in thylakoids obtained by either downshifting growth temperature or administration of benzyl alcohol was paralleled with enhanced thermosensitivity of the photosynthetic membrane. Simultaneously, the stress-sensing system leading to the cellular heat shock (HS) response also has been altered. There was a close correlation between thylakoid fluidity levels, monitored by steady-state 1,6-diphenyl-1,3,5-hexatriene anisotropy, and threshold temperatures required for maximal activation of all of the HS-inducible genes investigated, including dnaK, groESL, cpn60, and hsp17. The causal relationship between the pre-existing thylakoid physical order and temperature set point of both the transcriptional activation and the de novo protein synthesis was the most striking for the 17-kDa HS protein (HSP17) associated mostly with the thylakoid membranes. These findings together with the fact that the in vivo modulation of lipid saturation within cytoplasmic membrane had no effect on HS response suggest that thylakoid acts as a cellular thermometer where thermal stress is sensed and transduced into a cellular signal leading to the activation of HS genes.

Contents of childbirth-related fear among couples wishing the partner's presence at delivery.

In order to explore the contents of childbirth-related fears, a survey was carried out among 216 pairs of prospective parents who opted for the presence of the partner at delivery. Each couple took part in a three-class parentcraft course during the third trimester. During the first antenatal class, couples were asked to fill in a questionnaire with inquiries about specific contents of fear they might have in relation to pregnancy, childbirth and relationship with their partner after childbirth. Each item of the questionnaire called for an answer from a five-grade scale of fear such as 'absolutely not', 'slightly', 'quite', 'quite strongly' and 'very much'. Their worries were ranked according to the weighted average of the frequency of positive answers. More than 80% of both men and women had some fears relating to childbirth. Women were most worried about, in order of significance, having a malformed or injured baby, assisted or operative delivery, being lonely in a strange environment, doing something wrong, and facing the uncertainties of how the delivery was going to happen. The wife having severe pain and suffering, operative delivery, fetal birth injuries, helplessness, powerlessness and the wife's death in childbirth were the most significant subjects of men's fears. Eighty per cent of women and 76% of men felt that the presence of the partner at delivery would have no adverse effect on their future personal relationship.

Chaperonin genes of the Synechocystis PCC 6803 are differentially regulated under light-dark transition during heat stress.

Transcriptional startpoints of the two heat inducible chaperonin genes of Synechocystis PCC 6803 were mapped within the conservative CIRCE element and proved to be identical irrespective of the temperature treatment. Finding of an ORF encoding for a potential CIRCE binding repressor (HrcA) further suggests that both groEL-analogs are regulated in a CIRCE-dependent manner. In contrast to the expectations, the chaperonin twins are differentially expressed under light-dark transition during heat stress. Not the light per se, but rather the photosynthetic electron transport appears to be accountable for the regulatory differences. Our findings support the hypothesis that multiple chaperonins play different physiological roles under stress conditions.

Bimoclomol: a nontoxic, hydroxylamine derivative with stress protein-inducing activity and cytoprotective effects.

Preservation of the chemical architecture of a cell or of an organism under changing and perhaps stressful conditions is termed homeostasis. An integral feature of homeostasis is the rapid expression of genes whose products are specifically dedicated to protect cellular functions against stress. One of the best known mechanisms protecting cells from various stresses is the heat-shock response which results in the induction of the synthesis of heat-shock proteins (HSPs or stress proteins). A large body of information supports that stress proteins--many of them molecular chaperones--are crucial for the maintenance of cell integrity during normal growth as well as during pathophysiological conditions, and thus can be considered "homeostatic proteins." Recently emphasis is being placed on the potential use of these proteins in preventing and/or treating diseases. Therefore, it would be of great therapeutic benefit to discover compounds that are clinically safe yet able to induce the accumulation of HSPs in patients with chronic disorders such as diabetes mellitus, heart disease or kidney failure. Here we show that a novel cytoprotective hydroxylamine derivative, [2-hydroxy-3-(1-piperidinyl) propoxy]-3-pyridinecarboximidoil-chloride maleate, Bimoclomol, facilitates the formation of chaperone molecules in eukaryotic cells by inducing or amplifying expression of heat-shock genes. The cytoprotective effects observed under several experimental conditions, including a murine model of ischemia and wound healing in the diabetic rat, are likely mediated by the coordinate expression of all major HSPs. This nontoxic drug, which is under Phase II clinical trials, has enormous potential therapeutic applications.

Evidence for a lipochaperonin: association of active protein-folding GroESL oligomers with lipids can stabilize membranes under heat shock conditions.

During heat shock, structural changes in proteins and membranes may lead to cell death. While GroE and other chaperone proteins are involved in the prevention of stress-induced protein aggregation and in the recovery of protein structures, a mechanism for short-term membrane stabilization during stress remains to be established. We found that GroEL chaperonin can associate with model lipid membranes. Binding was apparently governed by the composition and the physical state of the host bilayer. Limited proteolysis of GroEL oligomers by proteinase K, which removes selectively the conserved glycine- and methionine-rich C terminus, leaving the chaperonin oligomer intact, prevented chaperonin association with lipid membranes. GroEL increased the lipid order in the liquid crystalline state, yet remained functional as a protein-folding chaperonin. This suggests that, during stress, chaperonins can assume the functions of assisting the folding of both soluble and membrane-associated proteins while concomitantly stabilizing lipid membranes.

Fluorescence detection of symmetric GroEL14(GroES7)2 heterooligomers involved in protein release during the chaperonin cycle.

The GroEL14 chaperonin from Escherichia coli was labeled with 5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid (I-AEDANS), a hydrophobic probe whose fluorescent emission is sensitive to structural changes within the protein. Increasing concentrations of ATP or adenylyl imidodiphosphate but not ADP caused two successive GroES7-dependent changes in the fluorescence intensity of AEDANS-GroEL14, corresponding to the sequential binding of two GroES7 heptamers and the formation of two types of chaperonin heterooligomers, GroEL14GroES7 and GroEL14(GroES7)2. The binding of thermally denatured malate dehydrogenase (MDH) caused a specific increase in fluorescence intensity of AEDANS-GroEL14 that allowed the direct measurement in solution at equilibrium of ATP- and GroES7-dependent protein release from the chaperonin. Structure/function analysis during the generation of ATP from ADP indicated the following sequence of events: 1) ADP-stabilized MDH-GroEL14GroES7 particles bind newly formed ATP. 2) MDH-GroEL14GroES7 particles bind a second GroES7. 3) MDH-GroEL14(GroES7)2 particles productively release MDH. 4) Released MDH completes folding. Therefore, the symmetrical GroEL14(GroES7)2 heterooligomer is an intermediate after the formation of which the protein substrate is productively released during the chaperonin-mediated protein folding cycle.

The N-terminal half of a mitochondrial presequence peptide inserts into cardiolipin-containing membranes. Consequences for the action of a transmembrane potential.

The orientation of a mitochondrial-presequence peptide, associated with anionic lipid-containing model membranes, was investigated. The peptide inserts with its N-terminal alpha-helical part into cardiolipin (CL) monolayers so that the N-terminal 14 residues are protected from proteinase K. In phosphatidylglycerol (PG) monolayers the inserted peptide was fully accessible to the protease. A consequence of the different orientations of the peptide was that membrane potential-dependent protection from trypsin was much faster for the peptide bound to PG-containing vesicles compared to CL-containing membranes, suggesting that in the mitochondrial protein import process other components of the import apparatus are involved in the efficient potential-driven translocation of presequences across the inner mitochondrial membranes.

[Unstable angina pectoris associated with surgically treated coronary artery atresia]. / Instabil angina pectorissal járó egyszájadékú koszorúér-hálózat operált esete.

A case report of a rare, presumably congenital form of "single coronary artery", is presented. The authors review the classification and clinical significance of the entity with special emphasis with regard to anginal symptoms and the so called sudden unexpected death syndrome. Details and result of surgical intervention (conventional aorto-coronary saphenous bypass) are discussed.

Brainstem auditory evoked potentials during a helium-oxygen saturation dive to 450 meters of seawater.

When divers are exposed to extreme atmospheric pressures they may exhibit symptoms of the high pressure nervous syndrome (HPNS). Although clinical HPNS symptoms are well described, little is known about the underlying pathophysiologic mechanisms. Special HPNS signs like vertigo and tremor suggested sensory-motor hyperexcitability resulting from brainstem dysfunction. We therefore studied brainstem auditory evoked potential (BAEP) repeatedly in four divers during an experimental deep helium-oxygen saturation dive to 450 meters of seawater (msw). Wave I (auditory nerve response) latency decreased whereas interpeak latencies (IPLs) I-III and I-V, which indicate respective cochleo-pontine and cochleo-mesencephalic transmission time, prolonged during the dive. IPLs III-V also prolonged the dive, but with greater variability among divers. Two divers showed a marked reversal of the normal attenuation effect of increased stimulus presentation rates on IV and V amplitudes during compression, an effect that subsided during the stay at bottom depth. This finding might indicate a relative enhancement of synaptic excitability and is presumed to be a feature of HPNS. Wave I latency reduction might at least partly be caused by accelerated sound conduction in dense helium. Additionally, an upward shift of middle ear resonance frequencies in helium can induce a basal shift of the main cochlear portion responding to the wide band clicks. This effect may reduce wave I latency due to greater relative input from the basal high frequency-short latency-cochlear neurons. Pressure-induced decrease of nerve conduction velocity, delay of synaptic transmission, and inhibitory modulation of midbrain auditory afferents possibly contributed to observed interpeak latency prolongations. Clinical HPNS signs, such as tiredness, dizziness, postural and intentional hand tremor, ataxia, and opsoclonus, were noted in three divers after reaching 300 msw and continued throughout the 37-h stay at bottom depth.

The full length of a mitochondrial presequence is required for efficient monolayer insertion and interbilayer contact formation.

The peptide specificity of both presequence-monolayer interactions and the ability of presequences to induce interbilayer contacts between large unilamellar vesicles was investigated. A range of different synthetic peptides that are documented for their mitochondrial protein import abilities were used for this purpose. Both monolayer insertion and vesicle aggregation were found to be strongly dependent on the primary structure of the studied presequence peptides. The combination of monolayer data and results of vesicle aggregation experiments leads to the overall suggestion that monolayer insertion and interbilayer contact formation are mechanistically related. For maximal effects the full length of a presequence peptide is required. The cardiolipin specificity of presequence-induced interbilayer contact formation previously reported was found to be a more general property among presequence peptides. The peptide's ability to induce vesicle-vesicle contacts seems to parallel the efficiency of its import ability into mitochondria. These results lead to an extended hypothesis on the role of presequence-induced contact site formation during the mitochondrial protein import process.

Presequence-mediated intermembrane contact formation and lipid flow. A model membrane study.

The ability of a synthetic peptide corresponding to the presequence of cytochrome c oxidase subunit IV from yeast to cause intermembrane contacts was investigated using monolayer techniques. The presequence inserted efficiently into the monolayer with a specificity for the mitochondrial cardiolipin. In the inserted form, the peptide strongly promoted the formation of close contacts with large unilamellar lipid vesicles present in the subphase, a property which was also specific for cardiolipin. The contacts formed were stable and tight and resulted in the flow of lipids from the vesicles to the monolayer. These results led to new suggestions on the involvement of intermembrane contact formation in mitochondrial protein import and membrane biogenesis.

Homogeneous catalytic deuteration of fatty acyl chains as a tool to detect lipid phase transitions in specific membrane domains: a Fourier Transform Infrared spectroscopic study.

Synthetic phospholipid molecules have been deuterated by using a water soluble catalyst and deuterium gas. The physical state of both deuterated segments and unaffected bulk part of the lipid molecules can be monitored simultaneously by Fourier Transform Infrared Spectroscopy. It is shown on multilamellar phospholipid systems that the deuterated segments can be used as structural probes. Whereas the nu(C-H) frequencies represent an average conformational order along all the alkyl chains present, by following changes in nu(C-D) vibrations, mobility of those membrane domains deuterium labeled at specific depths in the hydrocarbon core can be estimated. The potential importance of this new approach in the study of biological membranes is discussed.

[Synthesis of selegiline, its potential impurities and metabolites]. / Selegilin, valamint lehetséges szennyezéseinek és metabolitjainak elöállítása.

Selegiline is prepared in a three-step synthesis starting from phenylacetone and methylamine. Its possible impurities may be the (+)-isomer and compounds formed in side reactions. The syntheses of selegiline's metabolites are also described.

[Study of selegiline and related compounds with x-ray diffraction]. / Selegilin és rokon vegyületek vizsgálata röntgendiffrakcióval.

Selegiline and its parent compounds were studied by X-ray diffraction. It was established that the racemates of primary and secondary amines (p-fluoro-amphetamine, methamphetamine, p-fluoro-methamphetamine) hydrochloride do not form racemic compounds but crystalline as conglomerates, at the same time tertiary amines like selegiline and p-fluoro-selegiline hydrochlorides do. The crystalline structure of five enantiomeric hydrochlorides were determined, the CPhe-C-C-N torsion angle is anti-periplanar in all cases but in p-fluoro-amphetamine where it is gauche.

The pharmacology of 1-phenyl-2-propylamino-pentane (PPAP), a deprenyl-derived new spectrum psychostimulant.

The peculiar tyramine uptake inhibitory effect of (-)deprenyl prompted structure-activity relationship studies aiming to develop new spectrum central nervous system stimulants which are devoid of MAO inhibitory potency and operate de facto as indirectly acting, nonreleasing sympathomimetics. Of the derivatives synthesized for this purpose, 1-phenyl-2-propylaminopentane (PPAP) was selected as the reference substance and its pharmacological spectrum is presented. PPAP is taken up by the catecholamine axon terminal membrane and the vesicular membrane but it is devoid of catecholamine-releasing property. As a result, PPAP is, by interference, a potent inhibitor of the uptake of indirectly acting sympathomimetic releasers and of the catecholamine transmitters. This was proved, on the one hand, by measuring the uptake of [14C]PPAP into the catecholaminergic axon terminals and the inhibition of the uptake of [3H]noradrenaline and [3H]dopamine by PPAP in the rat brain, and, on the other hand, on the pulmonary artery strip of the rabbit and, in vivo, using the rat nictitating membrane as a detector. PPAP increases motility at 2 mg/kg and, in contrast to amphetamine, inhibits it at very high doses (50 mg/kg) only. A two-sided antagonism in the motility-increasing effect between PPAP and amphetamine and, more pronounced, between PPAP and mazindol was detected. PPAP is substantially less effective in inducing stereotyped behavior than either amphetamine or methamphetamine. PPAP facilitates learning and retention, is highly potent in antagonizing the tetrabenazine-induced depression in behavioral tests and is very effective in the forced swimming test. Whereas amphetamines facilitate performance in a very narrow range of low doses, which turns, at a modest elevation of the dose, into the opposite effect, PPAP improves performance within a reasonably broad dose range. Based on the peculiar pharmacological profile of PPAP, its potential usefulness in depression, in Alzheimer's disease and in attention-deficit-hyperkinetic disorder seems to be plausible.