[Negative symptoms of schizophrenic patients from the perspective of psychiatrists, patients themselves and their relatives]. / Negative Symptome schizophrener Patienten aus der Perspektive der Psychiater, der Patienten selbst und deren Angehörigen.

Clinical experience shows that negative symptoms are affected by environmental factors. Thus, different assessors with different information about patient behavior in different environments may come to different findings of negative symptoms. In this regard, the present study evaluates to what extent the assessment of negative symptoms by schizophrenic inpatients and their relatives compares to interview-based assessments by experts. Therefore, 33 schizophrenic patients were rated by patients themselves, their relatives, and psychiatrists. Negative symptoms were assessed with comparable assessment scales using the modified version of the Scale for the Assessment of Negative Symptoms (SANS) for patients or relatives and the original SANS for psychiatrists. Analyses revealed that the total SANS summary scores as rated by patients and relatives were comparable to scores rated by psychiatrists. Scores on SANS subscales of "alogia" and "attention deficits" differed significantly among the three ratings, while psychiatrists rated the patients' impairments as lower than did the patients themselves or their relatives. These findings indicate that patients' and relatives' ratings could be used to reduce information variance and improve the validity of interview-based, assessed negative symptoms.

Morphology-related effects on gene expression and protein accumulation of the yeast Arxula adeninivorans LS3.

The dimorphism of the yeast Arxula adeninivorans LS3 is regulated by cultivation temperatures. Up to 42 degrees C the yeast grows as budding cells, which turn to mycelia at higher temperatures. To test whether the dimorphism is exclusively induced by high temperatures or also by other conditions, mutants were selected with an altered behaviour with respect to dimorphism. After mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine, five of 25,000 colonies formed a very rough surface consisting of mycelia at 30 degrees C, in contrast to the wild-type. These mutants allow temperature-mediated and morphology-related effects on gene expression and protein accumulation to be distinguished. Budding cells and mycelia showed different expression of genes encoding secretory proteins at the same temperature. Mycelia secreted two-fold more protein than budding cells, including the enzymes glucoamylase and invertase. This indicated that morphology, rather than temperature, is the decisive factor in the analysed processes.

The MATA locus of the dimorphic yeast Yarrowia lipolytica consists of two divergently oriented genes.

The MATA locus of Yarrowia lipolytica, which was on the basis of its ability to induce sporulation in a diploid B/B strain, represses the mating capacity of this strain. The gene functions required for induction of sporulation and repression of conjugation could be separated by subcloning. Sequence analysis revealed two ORFs in the MATA locus. One of them (MATA1) codes for a protein of 119 amino acids which is required to induce sporulation. The other (MATA2) codes for a protein of 291 amino acids that is able to repress conjugation. Both genes are oriented divergently from a central promoter region, which possesses putative TATA and CAAT boxes for both genes. The product of MATA1 shows no homology to any known protein and seems to represent a new class of mating-type genes. MATA2 contains a HMG box with homology to other mating-type genes. Both MATA1 and MATA2 are mating-type specific. In cells of both mating types, the regions flanking the MATA locus contain sequences with homology to either S. cerevisiae SLA2 and ORF YBB9, respectively. From hybridization and subcloning data we estimate that the MATA region is approximately 2 kb long and is present only once in the genome.

The green fluorescent protein targets secretory proteins to the yeast vacuole

The green fluorescent protein (GFP) was used as a marker to study the intracellular transport of vacuolar and secretory proteins in yeast. Therefore, the following gene constructs were expressed in Saccharomyces cerevisiae under control of the GAL1 promoter: GFP N-terminally fused to the yeast secretory invertase (INV-GFP), the plant vacuolar chitinase (CHN-GFP) and its secretory derivative (CHNDeltaVTP-GFP), which did not contain the vacuolar targeting peptide (VTP), both chitinase forms (CHN and CHNDeltaVTP), GFP without any targeting information and two secretory GFP variants with and without the VTP of chitinase (N-GFP-V and N-GFP). Whereas chitinase without VTP is accumulated in the culture medium the other gene products are retained inside the cell up to 48 h of induction. Independently of a known VTP they are transported to the vacuole, so far as they contain a signal peptide for entering the endoplasmic reticulum. This was demonstrated by confocal laser scanning microscopy, immunocytochemical analysis and subcellular fractionation experiments as well. The transport of the GFP fusion proteins is temporary delayed by a transient accumulation in electron-dense structures very likely derived from the ER, because they also contain the ER chaperone Kar2p/Bip. Our results demonstrate that GFP directs secretory proteins without VTP to the yeast vacuole, possibly by the recognition of an unknown vacuolar signal and demonstrates, therefore, a first limitation for the application of GFP as a marker for the secretory pathway in yeast.

The peptide HDEF as a new retention signal is necessary and sufficient to direct proteins to the endoplasmic reticulum.

The key feature of tomato RNase LX localised solely outside the vacuole is the C-terminal peptide HDEF which is very similar to known endoplasmic reticulum (ER) retention signals. For functional testing of the ER-targeting ability of HDEF, different constructs including the complete RNase LX, two truncated forms without HDEF and the truncated chitinase FB7-1deltaVTP C-terminally flanked by HDEF, were expressed in Saccharomyces cerevisiae. The majority of RNase and chitinase, both containing HDEF, accumulates within the ER. However, the truncated constructs without the peptide are released into the medium. We provide compelling evidence that peptide HDEF at the C-terminus of secretory plant proteins is a new ER retention signal in yeast and most likely in plants.

AILV1 gene from the yeast Arxula adeninivorans LS3--a new selective transformation marker.

The ILV1 gene of the yeast Arxula adeninivorans LS3 (AILV1) has been cloned from a genomic library, characterized and used as an auxotrophic selection marker for transformation of plasmids into this yeast. One copy of the gene is present in the Arxula genome, comprising 1653 bp and encoding 550 amino acids of the threonine deaminase. The protein sequence is similar (60.55%) to that of the threonine deaminase from Saccharomyces cerevisiae encoded by the gene ILV1. The protein is enzymatically active during the whole period of cultivation, up to 70 h. Maximal activities, as well as protein concentrations of this enzyme, were achieved after cultivation times of 20-36 h. The AILV1 gene is a suitable auxotrophic selection marker in transformation experiments using an Arxula adeninivorans ilv1 mutant and a plasmid containing this gene, which is fused into the 25S rDNA of Arxula adeninivorans. One to three copies of the linearized plasmid were integrated into the 25S rDNA by homologous recombination. Transformants resulting from complementation of the ilv1 mutation can be easily and reproducibly selected and in addition are mitotically stable. Therefore, the described system is preferred to the conventional selection for hygromycin B resistance.

Humoral immune response to a 200-kDa glycoprotein antigen of Saccharomyces cerevisiae is common in man.

According to Heelan et al. patients suffering from Crohn's disease (CD) produce antibodies against a cell wall associated glycoprotein antigen gp200 of the yeast Saccharomyces cerevisiae, while healthy people do not. Here the authors show, that antibodies against this glycoprotein gp200 can also be detected in the sera of healthy humans. The intensity of the antibody titer which is measured by immunoblot experiments is independent from the state of health. The Saccharomyces cerevisiae specific gp200 is a highly glycosylated protein localized not only in the cell wall but also accumulated in the culture medium. Some of the tested sera from CD patients, as well as from healthy adults, also reacted with a 120-kDa glycoprotein which is to be found in preparations containing secreted proteins. Because the binding of antibodies is greatly reduced by periodate treatment of gp200 and by the 120-kDa polypeptide, it is very likely that their carbohydrate moieties are the antigenic determinants against which the specific human antibodies are directed. The human humoral immune response applies only to Saccharomyces cerevisiae antigens, because no analogous immune responses could be detected against antigens derived from the yeast Arxula adeninivorans.

Evidence for secretion of vacuolar alpha-mannosidase, class I chitinase, and class I beta-1,3-glucanase in suspension cultures of tobacco cells.

We have investigated the possibility that vacuolar proteins can be secreted into the medium of cultured cells of Nicotiana tabacum L. Time-course and balance-sheet experiments showed that a large fraction, up to ca. 19%, of vacuolar alpha-mannosidase (EC 3.2.1.24) and vacuolar class I chitinase (EC 3.2.1.14) in suspension cultures accumulated in the medium within one week after subculturing. This effect was most pronounced in media containing 2,4-dichlorophenoxyacetic acid (2,4-D). Under comparable conditions only a small fraction, 1.8-5.1% of the total protein and ca. 1% of malate dehydrogenase (EC 1.1.1.37), which is localized primarily in the mitochondria and cytoplasm, accumulated in the medium. Pulse-chase experiments showed that newly synthesized vacuolar class I isoforms of chitinase and beta-1,3-glucanase (EC 3.2.1.39) were released into the medium. Post-translational processing, but not the release of these proteins, was delayed by the secretion inhibitor brefeldin A. Only forms of the proteins present in the vacuole, i.e. mature chitinase and pro-beta-1,3-glucanase and mature beta-1,3-glucanase, were chased into the medium of tobacco cell-suspension cultures. Our results provide strong evidence that vacuolar alpha-mannosidase, chitinase and beta-1,3-glucanase can be secreted into the medium. They also suggest that secretion of chitinase and beta-1,3-glucanase might be via a novel pathway in which the proteins pass through the vacuolar compartment.

Correct targeting of a vacuolar tobacco chitinase in Saccharomyces cerevisiae--post-translational modifications are dependent on the host strain.

The chitinase gene FB7-1 of Nicotiana tabacum cv. samsun line 5 was expressed in the two Saccharomyces cerevisiae strains, INVSC2 and H4, under the control of the GAL1 promoter from S. cerevisiae and a multicopy plasmid vector. Both yeast strains express the plant gene as enzymatic active proteins. In transformants of the strain INVSC2, 94% of the total plant chitinase is contained inside the cells, probably within the vacuole which has been confirmed by subcellular fractionation as well as immunohistochemical experiments. This retention inside the cells is due to the C-terminally located 7 amino acids long vacuolar targeting peptide of the prochitinase. When this sequence was removed, chitinase was transported into the culture medium. Pulse-chase experiments revealed that during translation in transformants of both yeast strains one chitinase polypeptide can be immunoadsorbed with specific antibodies. In the case of INVSC2-transformants, newly formed chitinase is modified in a 60 min chase to slightly increase its molecular mass, whereas in H4-transformants the molecular mass constantly remained 32 kDa. By Western blot analysis two chitinase corresponding polypeptides of 32 and 37 kDa were accumulated in the culture medium of both transformants carrying the chitinase gene without the vacuolar targeting sequence. The larger one was very likely O-glycosylated. Whereas, both polypepitdes were also detected in cell extracts of the H4-transformant, only the smaller one was found in the INVSC2-transformant. The plant chitinase passed through the endoplasmic reticulum on its way to the vacuole. The N-terminal signal peptide responsible for the uptake into the endoplasmic reticulum is cleaved correctly. However, cleavage of the vacuolar targeting peptide located at the C-terminus, to give the mature chitinase is obviously influenced by the genetic background of the host strain. In INVSC2-transformants chitinase accumulates in its mature form whereas both the polypeptides of H4-transformants retain their vacuolar targeting peptide. Our results demonstrate that in the case of plant class I chitinase, the plant sorting signal is recognized in yeast cells but post-translational modifications are influenced by the host strain.

Long-term effects of restrictive culture conditions on Saccharomyces cerevisiae sec7 cells.

Long-term effects of restrictive conditions on the temperature-sensitive S. cerevisiae sec7 mutant were studied. By microscopic analysis no cell lysis could be detected of cells cultured for up to 19 days at 37 degrees C. The optical density as well as the cell number remained constant during the whole period under restrictive conditions. However, restrictive conditions decreased the incorporation of 35S-methionine into intracellular proteins in a reversible manner indicating that protein biosynthesis was inhibited whereas the cells remained alive. Northern blot experiments revealed that restrictive conditions did not markedly decrease the ratio of the mRNA levels to total RNA for the genes TEF1, TEF2, SUC2, and BGL2 up to 73 hours. However the content of total RNA decreased drastically with increasing incubation times at restrictive temperature. In spite of the reduced total RNA content, cells are capable of new synthesis of mRNA under restrictive conditions which was shown by incubation of the cells in the presence of actinomycin D--an inhibitor of the mRNA synthesis. Most of the cells which survived a long-term incubation at 37 degrees C are not able to divide and to form colonies immediately after their transfer to permissive conditions.

Expression of the Arxula adeninivorans glucoamylase gene in Kluyveromyces lactis.

The glucoamylase gene of the yeast Arxula adeninivorans was expressed in Kluyveromyces lactis by using the GAP promoter from Saccharomyces cerevisiae and a multicopy plasmid vector. The transformants secreted 90.1% of the synthesized glucoamylase into the culture medium. The secreted glucoamylase activities are about 20 times higher in comparison to those of Saccharomyces cerevisiae transformants using the same promoter. Secreted glucoamylase possesses identical N-terminal amino acid sequences to those secreted by A. adeninivorans showing that cleavage of the N-terminal signal peptide takes place at the same site. Biochemical characteristics of glucoamylase expressed by K. lactis and A. adeninivorans are very similar.

Temperature-dependent dimorphism of the yeast Arxula adeninivorans Ls3.

Arxula adeninivorans Ls3 is described as an ascomycetous, arthroconidial, anamorphic, xerotolerant yeast, which was selected from wood hydrolysates in Siberia. By using minimal salt medium or yeast-extract-peptone-medium with glucose or maltose as carbon source it was shown that this yeast is able to grow at up to 48 degrees C. Increasing temperatures induce changes in morphology from the yeast phase to mycelia depending on an altered programme of gene expression. This dimorphism is an environmentally conditioned (reversible) event and the mycelia can be induced at a cultivation temperature of 45 degrees C. Depending on the morphology of strain Ls3 (yeast phase or mycelia) the secretion behaviour as well as the spectrum of polypeptides accumulated in the culture medium changed. The activities of the accumulated extracellular enzymes glucoamylase and invertase were 2 to 3 times higher in cultures grown at 45 degrees C than in those grown at 30 degrees C. While the level of the glucoamylase protein secreted from mycelia between 45 and 70 hours did not change, biochemical activity decreased after a cultivation time of 43 hours. It was shown that this effect depended on both the catabolic repression of the glucoamylase by glucose and the thermal inactivation of this enzyme in media without or with low concentrations of starch or maltose.

Comparative biochemical, genetical and immunological studies of glucoamylase producing Arxula adeninivorans yeast strains.

Seven Arxula adeninivorans strains originating from The Netherlands (CSIR 1136, CSIR 1138 and CBS 8244T), South Africa (CSIR 1147, CSIR 1148 and CSIR 1149) and Siberia (Ls3) were compared concerning their secretory glucoamylase. Within the spectrum of secretory polypeptides obtained by SDS-polyacrylamide gel electrophoresis, the glucoamylase corresponding polypeptide, could be identified by means of specific antibodies directed against the glucoamylase of strain Ls3. The molecular masses of the glucoamylases vary from 84 to 95 kDa. After endoglycosidase F treatment of the secretory proteins, the N-linked carbohydrate content for each glucoamylase could be quantified at about 25%. Glucoamylase activity staining of secretory proteins separated under nondenaturing conditions revealed one glucoamylase active protein for the Dutch strains and two active forms for the strains originating from South Africa and Siberia. Highest activities of glucoamylase can be measured at the end of the exponential growth phase for each strain. The Dutch strain CSIR 1138 secretes the highest activity. Optimum values for temperature and pH were determined and compared. By means of pulsed field gel electrophoresis and Southern analysis, the glucoamylase corresponding gene could be localized on the second of the four chromosomes of each yeast strain.

Characterization of Arxula adeninivorans strains from different habitats.

Some Arxula adeninivorans strains selected from wood hydrolysates in Siberia, from soil in South Africa and from maize silage and soil in The Netherlands were compared. DNA-fingerprinting, pulse field gel electrophoresis as well as analysis of secretory proteins have been chosen to describe the similarities among the strains. Combination of the three methods allowed identification of each strain. Strains from the same origin show extensive similarities. The results of the DNA-fingerprints indicate that the strain isolated in Siberia belongs to the group of strains originated from South Africa. However, it differed in the molecular weight of the third chromosome and in the pattern of secretory proteins from the South African isolates.

[Heparin therapy in embolic cerebral infarct. A retrospective study]. / Heparintherapie bei embolischen, zerebralen Insulten. Eine retrospektive Untersuchung.

Heparin therapy in acute stroke is a controversial issue. It is uncertain, whether heparin has a therapeutic or preventive effect in the early phase of the stroke. From 1984-1989, 1095 patients with acute ischemic stroke were treated, 141 (12.9%) of whom received heparin within 3 days of stroke onset. The mean duration of heparin anticoagulation was 10 days. In 28 cases (20%), heparin was used as antithrombotic agent (25/28 patients suffered a basilar artery occlusion, of whom 22 died). In 113 cases (80%), heparin was used in embolic stroke to prevent recurrence (24% cardioembolic stroke, 54% arterio-arterial embolism, and 22% embolism of unknown etiology). The rate of recurrent stroke in the early phase was 13% with a persistent deficit in 5.3%. The results are comparable with those of other trials reported in the literature. Only 2 patients had an anticoagulation-related haemorrhage with clinical deterioration. Heparin anticoagulation in acute stroke is a low-risk therapy, but its preventive value has not yet been demonstrated.

[Heart wall rupture as a grave complication of acute myocardial infarct]. / Die Herzwandruptur als schwerwiegende Komplikation des akuten Herzinfarktes.

The rupture of the heart wall is a severe complication of the acute myocardial infarction. We found it in 3.5% of the deceased patients with an acute myocardial infarction. The average age of these patients was 71 years. 75% of the patients died during the first five days after the event of the myocardial infarction. Apart from elderly patients with myocardial infarction such ones with a transmural myocardial infarction in the region of the left ventricle, an enlargement of the heart and signs of an insufficiency of the left heart, with a hypertension and diabetes mellitus seemed to be endangered. These patients need the most exact control and observation and in case of suspicion (symptomatology of angina pectoris which is continuing to exist) of a developing rupture of the heart wall and aimed diagnostics (echocardiography) and therapy must be begun immediately.

Concomitant changes of membrane leak permeability and phospholipid dynamics in erythrocytes subjected to chemical and physical membrane perturbation.

A mild selective oxidant of erythrocyte membrane SH-groups producing SS-bonds and inducing reversible crosslinking of spectrin, as well as strong less specific oxidants which produce a more extensive modification of membrane proteins and peroxidation of lipids and dielectric breakdown of the membrane induce formation of structural defects acting as aqueous pores and reorientation sites for phospholipids. The coupling of a process requiring hydrophilic structures (leak permeability) and a process also involving hydrophobic constituents (flip of phospholipids) suggests that the membrane lipid domain is involved in the effects.

Transbilayer mobility of phospholipids in the erythrocyte membrane. Influence of the membrane skeleton.

Oxidative treatment of erythrocytes results in a strong enhancement of transbilayer reorientation of exogenous lysophospholipids. Only upon selective oxidation of SH-groups and concomitant crosslinking of spectrin by diamide, however, asymmetry of distribution of inner layer phospholipids, phosphatidylethanolamine and phosphatidylserine in the erythrocyte membrane becomes lost. This indicates that asymmetry is not due to a low transbilayer reorientation of the inner layer phospholipids, but that phosphatidylethanolamine and phosphatidylserine do not have access to the flip sites in the native membrane. A role of membrane skeleton proteins in the suppression of access of inner layer phospholipids to the flip sites is indicated by the loss of asymmetry of both phospholipids in spectrin depleted inside out vesicles and in vesicles produced by heat denaturation of spectrin. This idea is further supported by the observation that inner layer phospholipids analogues lysophosphatidylethanolamine and lysophosphatidylserine spontaneously accumulate in the inner layer of native erythrocytes, whereas lysolecithin does not. Moreover, this asymmetry is abolished in inside out vesicles.

[Computer-assisted documentation in anesthesia (author's transl)]. / Autonome rechnerunterstützte Anästhesiedokumentation. Erfahrungen nach zweijährigem Einsatz.

Details are given of a Dietz microprocessor with 48 K Bytes core memory for anaesthetic documentation. The experiences gained with this equipment over a period of two years are reviewed. The storage capacity is sufficiently large to allow its use by several persons for the processing of more than 20,000 anaesthetic records per year. The preparation of the anaesthetic record takes a skilled technician about two minutes. The prompt evaluation of the data and their presentation on either screen or print-out are the main advantages of this system. Information regarding the course of anaesthesia, complications, the identity of the anaesthetist are available instantly and at any time and can be referred to if organizational problems arise. The fact that all relevant data and criteria can be called up whenever wanted also acts as a spur to make use of the information. The system still needs some improvement to make it more efficient.