Regio entorhinalis in schizophrenia: more evidence for migrational disturbances and suggestions for a new biological hypothesis.

Recently we were able to replicate the original finding of migrational disturbances in the entorhinal cortex (ERC) of schizophrenic patients by measuring the distance of pre-alpha cell clusters to the pial surface. In order to replicate this finding, we performed a detailed analysis of the pre-alpha cell clusters in the ERC in post mortem brains of 22 schizophrenic patients and 15 control subjects. Cluster position relative to gray/white matter boundary were measured and normalized by the widths of the gray matter. In the ERC the pre-alpha cell clusters were situated significantly closer to the gray/white matter junction compared to normal controls (around 30 %, F = 9.52, p = 0.004). No specific effects of sex, age or region of investigation were found. In summary, this is another quantitative replication of pre-alpha cell cluster migrational disturbances in schizophrenia, which are possibly linked to neurobiological abnormalities, e.g. myeloarchitectonic changes. This supports the notion that developmental abnormalities are a core feature of schizophrenia and that the search for candidate genes has to include this aspect, too. However, it is very probable that vulnerability-associated changes - as outlined here - have to be distinguished from disease-related changes.

Disturbed planum temporale asymmetry in schizophrenia. A quantitative post-mortem study.

The planum temporale of the temporal cortex was investigated post-mortem in 24 schizophrenic patients and 24 age- and sex-matched control subjects. Schizophrenic patients demonstrated a 20% volume reduction of the left planum temporale (p = 0.032), whereas on the right side, there was a trend for increase in male schizophrenics (+22%, p = 0.17), while in female patients the volume was moderately decreased (-6%, p = 0.74). The mean anterior-posterior diameter of the planum temporale was significantly reduced in the left hemisphere (-20%, p = 0.008), but unchanged on the right side. The asymmetry coefficients (Galaburda et al. (1987) Neuropsychologia 25, 853-868) for the planum temporale cortex volume (p = 0.02) and anterior-posterior diameter (p = 0.002) but not for mean area (p = 0.61) were significantly different between schizophrenics and control subjects. These data support the idea of disturbed cerebral laterality in schizophrenia. The implications of methodology and patient samples are discussed.

Loss of sylvian fissure asymmetry in schizophrenia. A quantitative post mortem study.

The sylvian fissure is known to be one of the most asymmetric structures of the human brain. Sylvian fissure length was measured in post-mortem brains of 35 schizophrenic patients and 33 matched non psychiatric control subjects. The schizophrenics showed a significantly reduced length of the left sylvian fissure (-16%, p less than 0.0001) compared to the control subjects, while the right sylvian fissure length was unchanged. Sylvian fissure asymmetry (left/right ratio) was more reduced in male schizophrenics (-24%, p less than 0.001) than in female patients (-16%, p less than 0.03). This finding is consistent with several post-mortem and MRI studies showing left temporal lobe pathology in a significant proportion of patients and may indicate that schizophrenia is a disorder of early neurodevelopment causing impaired cerebral lateralization.

Heterogeneity, distribution and endogenous phosphate content of KCl-myosins prepared from different parts of human brain.

Myosin concentrations and their endogenous phosphate (P) content as well as some of their properties were investigated in different parts of the central nervous system (CNS). Myosin concentration in hemispherial part is in the average 40 mg/100 g fresh mince, but it varies between 5 mg and 30 mg myosin depending on the mental activity or accidental brain diseases. This refers to the other parts of CNS, too. A myosin may be isolated with a single and sharp peak as far as the gel filtration profile is concerned in spite of the high lipid content of the brain. Working with minimal lipid and RNA content requires a careful procedure by an experienced person. In spite of any careful isolation procedure, purified KCl-myosin preparations are composed of at least two isomyosins each formed from different heavy chains. Myosins are localized in different parts of the cell, mitochondrial, synaptosomal and neuroplasmic, respectively. Brain myosins have higher endogeneous P concentrations than other cell-myosins. In hemispherial myosins the P-concentrations are high. Subjects trained in mental activities have the highest myosin and P concentration. We suggest that the role of myosin may directly be connected with cytokinetic motions even in case of the higher functions of brain cells like thinking, paying attention and memory, respectively.

Presence of phospho-tyrosine in alkaline hydrolysate of pig skeletal muscle myosin.

A small part of the P-containing fraction of myosin hydrolysate which could be eluted with basic solvent (triethylamine) buffer was accumulated on chromatographic column. On the basis of the elution profile, P- and tyrosine content determination, moreover the thin layer chromatographic separation and specific Tyr reaction, this fraction is suggested to be phosphotyrosine. The concentration of P-Tyr is about 2 mol in pig muscle myosin calculated for 500 kDa protein. As the higher P-containing myosins also have an approx. 2 mol P-Tyr, it is thought that this concentration had been present in myosins already before alkaline hydrolysis.

Presence of covalently bound energy-rich phosphates in human tracheal smooth muscle myosin.

In a preliminary report, the tracheal NaCl-myosin prepared from an old and a young subject was discussed. In the present paper, the total bound phosphate (P) content and its distribution is described in two parallel preparations of human muscle myosin. It was shown that a considerable amount of covalently bound P was present not only in NaCl, but in the fresh preparations of tracheal KCl-myosin. Analysing this phosphate fraction in the alkaline hydrolysate of RNA- and lipid-free preparations of myosin it was confirmed that phosphate was linked to the basic amino acid residues and their hydrolytic derivatives. As the phosphoryl binding sites are partly saturated, the phosphate concentration can be enhanced nearly three-fold compared to the fresh preparation. Phosphate incorporation is an autophosphorylation process depending on the ATP and Mg2+ concentration. Studying the actomyosin fraction in the presence of ATP it was found that its phosphate content can also be increased to a certain degree. It is supposed that the changes in phosphate content of myosin are associated with the formation of crossbridges between the actin and myosin filaments in the processes of muscle contraction and relaxation. The process can be influenced directly and indirectly by some natural factors and drugs resulting in the concentration or relaxation of bronchial muscles.

Composition of amino acid phosphates in phosphorylated G-actin of rabbit skeletal muscle.

In our experiments the phosphorylation of actin was studied. Similar investigations have been published in the literature, however very long incubation time was applied in these studies and even so a low incorporation of phosphate concentration was found. The present phosphorylation experiments were performed using short incubation periods as usual in our myosin investigations and was characterized by an unexpectedly high phosphate saturation. We suggest that in suitable incubation medium the nucleotide- and phosphate-free actin prepared by using phosphate- and ATP-free solutions takes only 1 minute to become saturated, while in its peptide chain a N-P bond type acid labile phosphate is formed. On maximum saturation 9 M P-arginine, 0.4 M P-histidine and some minor phosphorylated derivatives can be observed. After a longer period of incubation, with a lower incorporation of phosphate (3 mol P) a more stable phosphorylated actin is formed. As a result of preparation and gel filtration a dimer and a monomer form of actin can be obtained. Both of them exhibit the basic properties of actin (polymerization, myosin-ATPase activation) and the phosphate incorporation described in this paper.

Purification and properties of myosin from the "hatching muscle" (m. complexus) of geese.

The present work is concerned with the study of myosin fractions prepared from the hatching muscle (m. complexus) and a control muscle (m. pectoralis) of the developing goose embryo. The m. complexus attained its maximum mass at hatching and in the 4-day-old bird the mass of this muscle was only one fourth of that recorded at hatching. The m. complexus was hypertrophied already on the 21st day. At days 21, 27 and 28 of incubation and at posthatching days myosin preparations were made from both muscles. Partial purification of myosins from both sources yielded a high molecular weight fraction characteristic of the adult bird and one other protein fraction with molecular mass half of myosin. Both preparations exhibited the characteristic properties of myosin. The lower molecular weight fraction was also shown to develop filamentous aggregates as did the higher molecular-weight, gel filtrated myosin. The phosphate content of the half molecular mass myosin fraction prepared from the embryonic m. complexus at days prior to hatching was considerably higher than that of the high molecular weight fraction and the predominant component was P-Arg. Since the embryonic myosin was still not available in the m. complexus of the 4-day-old birds and the hypertrophied muscle underwent regression after hatching it appears that this myosin fraction is actively involved in breaking through the shell during the hatching period in geese.

Preparation and characterization of myosin from cultured cells of Escherichia coli (02:K1 30156 strain).

In this study the myosin preparations isolated from E. Coli cell cultures were analysed. The isolation of myosin from fresh cultures resulted in a substantially higher (approximately 10-fold) yield than from stored cells. The coli myosin, despite the two DEAE-cellulose treatments, contained more RNA and P-lipid than the myosin prepared from skeletal muscle. The RNA content can be removed gradually by acetone denaturation and lipid removal followed by subsequent washings. The fresh preparations contained protein-bound alkali-stable P; part of this was released by Cu ions. The coli myosin can be phosphorylated. The phosphate uptake depended on the concentrations of ATP and Mg2+ and on the time of incubation. The alkaline hydrolysate of the phosphorylated, washed lipid-free preparation was resolved into 8 P-containing peaks on ion-exchange chromatography. Of fractions P-Arg, Pi, P-Lys and 2 P-His conformer were identified by means of synthetic compounds, elution pattern and specific reactions. The remaining compounds could not be identified. The most abundant component was P-Arg suggesting that this compound might play an important role in the cytokinetic movements of E. coli.

Examination of a spontaneously transformed aortic smooth muscle cell line. I. Morphological examinations.

Spontaneous transformation was observed of a mini-pig aortic smooth muscle cell line in its 21st passage in vitro. The transformed cells exhibited changed phenotype, heteroploidy and excessive proliferative activity. The initially elongated cells developed a polymorphous essentially polygonal appearance. The longitudinal filaments characteristic of normal cells disappeared and only finely granular double refracting matter was detectable in polarized light. The transformed cells exhibited microvilli in both transmission electron microscopic and scanning electronmicroscopic preparations. The appearance of spontaneous lectin binding in the transformed cells is a sign of altered plasma membrane structure. The possible contaminational origin of the transformed cells was carefully excluded.

Isolation, properties and P content of the human brain myosin.

KCl-, and NaCl-myosins were prepared from different parts of the central nervous system (CNS). Throughout these experiments P and lipid contents were higher in NaCl-myosins than in KCl-preparations. Both KCl-, and NaCl-myosins have increased lipid and P contents compared with skeletal muscle myosins. When the specimens were separated by a molecular sieve, it was found by chromatographic technique on Sepharose 4B column that the cerebral and cerebellar myosins were composed of two fractions of different molecular mass while the brain stem and spinal cord myosins revealed only a single peak. The myosin fractions' Ca-ATPase activity could be augmented by rabbit muscle actin. The myosin preparations developed filamentous systems and aggregates which could be shown by scanning electron microscopy. All the CNS-myosin preparations could be phosphorylated; however, they were saturated to a different degree and were influenced by the presence or absence of serotonin. The kinetic studies revealed that the phosphate saturation of the brain stem, cerebellar and cerebral myosins depended on the ATP concentration and incubation time. The alkaline hydrolysates of lipid-free human brain myosin preparations contained amino acid phosphates, P-Arg, P-Lys and P-His in different amounts depending on their sources. In response to a phosphorylating mixture only the amount of P-Arg was elevated in the cerebral myosins, P-Arg and P-His in the brain stem preparations, and P-Arg, P-His and the amounts of unidentified compounds in the cerebellar ones.

Preparation and purification of myosin from human tracheal smooth muscle.

NaCl myosin was prepared from the annular smooth muscles of human bronchus. About 7 mg of gel filtered myosin was gained from 8 g minced tracheal muscle of the younger subject. The yield from the older (74-year old) subject was only 30% of that from the younger subject, even though the starting material was more (12 g minced tissue). Tracheal myosin contains P lipid in considerable amount; P lipids account for some 28% of the total phosphate content of the myosin, and even more (50-55%) in the case of the older subject. The preparation could be phosphorylated only in the presence of CAMP and PGF2 alpha, respectively. Cu2+ treatment liberated less phosphate when compared with myosin preparations from other smooth muscles; however, the majority of the phosphate bonds underwent hydrolysis upon the effect of KOH. The reactions specific for amino acids, and also other observations allow the conclusion that the majority of covalently bound phosphate is present in an ester-type bond. Lysine-vasopressin, and also diethylpyrocarbonate successfully protect the P content of myosin from the hydrolysis inherent to incubation.