Effect of intravenous low-intensity laser irradiation of the blood on clinical and laboratory parameters of hepatocellular insufficiency.

Patients with hepatocellular insufficiency received a course of intravenous laser irradiation of the blood. After the treatment, a positive dynamics of clinical and biochemical indices of the major hepatic syndromes was observed: alleviation of the major clinical symptoms and significant positive changes in biochemical parameters (AST, ALT, bilirubin, alkaline phosphatase, lactate dehydrogenase, and total cholesterol).

Abnormalities in Cortical GABAergic Interneurons of the Primary Motor Cortex Caused by Lis1 (Pafah1b1) Mutation Produce a Non-drastic Functional Phenotype.

LIS1 (PAFAH1B1) plays a major role in the developing cerebral cortex, and haploinsufficient mutations cause human lissencephaly type 1. We have studied morphological and functional properties of the cerebral cortex of mutant mice harboring a deletion in the first exon of the mouse Lis1 (Pafah1b1) gene, which encodes for the LisH domain. The Lis1/sLis1 animals had an overall unaltered cortical structure but showed an abnormal distribution of cortical GABAergic interneurons (those expressing calbindin, calretinin, or parvalbumin), which mainly accumulated in the deep neocortical layers. Interestingly, the study of the oscillatory activity revealed an apparent inability of the cortical circuits to produce correct activity patterns. Moreover, the fast spiking (FS) inhibitory GABAergic interneurons exhibited several abnormalities regarding the size of the action potentials, the threshold for spike firing, the time course of the action potential after-hyperpolarization (AHP), the firing frequency, and the frequency and peak amplitude of spontaneous excitatory postsynaptic currents (sEPSC's). These morphological and functional alterations in the cortical inhibitory system characterize the Lis1/sLis1 mouse as a model of mild lissencephaly, showing a phenotype less drastic than the typical phenotype attributed to classical lissencephaly. Therefore, the results described in the present manuscript corroborate the idea that mutations in some regions of the Lis1 gene can produce phenotypes more similar to those typically described in schizophrenic and autistic patients and animal models.

Doublecortin is a developmentally regulated, microtubule-associated protein expressed in migrating and differentiating neurons.

Recently, we and others reported that the doublecortin gene is responsible for X-linked lissencephaly and subcortical laminar heterotopia. Here, we show that Doublecortin is expressed in the brain throughout the period of corticogenesis in migrating and differentiating neurons. Immunohistochemical studies show its localization in the soma and leading processes of tangentially migrating neurons, and a strong axonal labeling is observed in differentiating neurons. In cultured neurons, Doublecortin expression is highest in the distal parts of developing processes. We demonstrate by sedimentation and microscopy studies that Doublecortin is associated with microtubules (MTs) and postulate that it is a novel MAP. Our data suggest that the cortical dysgeneses associated with the loss of Doublecortin function might result from abnormal cytoskeletal dynamics in neuronal cell development.

The lissencephaly gene product Lis1, a protein involved in neuronal migration, interacts with a nuclear movement protein, NudC.

Important clues to how the mammalian cerebral cortex develops are provided by the analysis of genetic diseases that cause cortical malformations [1-5]. People with Miller-Dieker syndrome (MDS) or isolated lissencephaly sequence (ILS) have a hemizygous deletion or mutation in the LIS1 gene [3,6]; both conditions are characterized by a smooth cerebral surface, a thickened cortex with four abnormal layers, and misplaced neurons [7,8]. LIS1 is highly expressed in the ventricular zone and the cortical plate [9,10], and its product, Lis1, has seven WD repeats [3]; several proteins with such repeats have been shown to interact with other polypeptides, giving rise to multiprotein complexes [11]. Lis1 copurifies with platelet-activating factor acetylhydrolase subunits alpha 1 and alpha 2 [12], and with tubulin; it also reduces microtubule catastrophe events in vitro [13]. We used a yeast two-hybrid screen to isolate new Lis1-interacting proteins and found a mammalian ortholog of NudC, a protein required for nuclear movement in Aspergillus nidulans [14]. The specificity of the mammalian NudC-Lis1 interaction was demonstrated by protein-protein interaction assays in vitro and by co-immunoprecipitation from mouse brain extracts. In addition, the murine mNudC and mLis1 genes are coexpressed in the ventricular zone of the forebrain and in the cortical plate. The interaction of Lis1 with NudC, in conjunction with the MDS and ILS phenotypes, raises the possibility that nuclear movement in the ventricular zone is tied to the specification of neuronal fates and thus to cortical architecture.

Variations in genes regulating neuronal migration predict reduced prefrontal cognition in schizophrenia and bipolar subjects from mediterranean Spain: a preliminary study.

Both neural development and prefrontal cortex function are known to be abnormal in schizophrenia and bipolar disorder. In order to test the hypothesis that these features may be related with genes that regulate neuronal migration, we analyzed two genomic regions: the lissencephaly critical region (chromosome 17p) encompassing the LIS1 gene and which is involved in human lissencephaly; and the genes related to the platelet-activating-factor, functionally related to LIS1, in 52 schizophrenic patients, 36 bipolar I patients and 65 normal control subjects. In addition, all patients and the 25 control subjects completed a neuropsychological battery. Thirteen (14.8%) patients showed genetic variations in either two markers related with lissencephaly or in the platelet-activating-factor receptor gene. These patients performed significantly worse in the Wisconsin Card Sorting Test-Perseverative Errors in comparison with patients with no lissencephaly critical region/platelet-activating-factor receptor variations. The presence of lissencephaly critical region/platelet-activating-factor receptor variations was parametrically related to perseverative errors, and this accounted for 17% of the variance (P = 0.0001). Finally, logistic regression showed that poor Wisconsin Card Sorting Test-Perseverative Errors performance was the only predictor of belonging to the positive lissencephaly critical region/platelet-activating-factor receptor group. These preliminary findings suggest that the variations in genes involved in neuronal migration predict the severity of the prefrontal cognitive deficits in both disorders.

The unfolding story of two lissencephaly genes and brain development.

Formation of our highly structured human brain involves a cascade of events, including differentiation, fate determination, and migration of neural precursors. In humans, unlike many other organisms, the cerebral cortex is the largest component of the brain. As in other mammals, the human cerebral cortex is located on the surface of the telencephalon and generally consists of six layers that are formed in an orderly fashion. During neuronal development, newly born neurons, moving in a radial direction, must migrate through previously formed layers to reach their proper cortical position. This is one of several neuronal migration routes that takes place in the developing brain; other modes of migration are tangential. Abnormal neuronal migration may in turn result in abnormal development of the cortical layers and deleterious consequences, such as Lissencephaly. Lissencephaly, a severe brain malformation, can be caused by mutations in one of two known genes: LIS1 and doublecortin (DCX). Recent in vitro and in vivo studies, report on possible functions for these gene products.

Differential expression of the human glucocerebrosidase-coding gene.

Gaucher disease is an inborn error of sphingolipid metabolism. It is due to decreased enzymatic activity of glucocerebrosidase (GCase) which causes accumulation of glucocerebrosides, mainly in cells of the reticulo-endothelial system. The disorder is clinically heterogenous and can include central nervous system signs. However, the manifestations of the disease in most cases are restricted to a limited number of cell types and organs. This could be explained by highly differential expression of the human gcs gene. To test this notion, the level of GCase-specific mRNA was determined in different human cell lines by hybridizing Northern blots to a human GCase-specific cDNA probe or by using the RNase protection method. It was found that epithelial cells exhibit high levels of GCase mRNA while skin fibroblasts and promyelocytes show intermediate steady-state levels of this RNA. Macrophages have low steady-state levels of GCase mRNA and in B-cells it is hardly detectable. Moreover, when B-cells or skin fibroblasts were transfected with a vector harbouring the bacterial cat gene coupled to the human gcs gene promoter, the levels of CAT expressed in each cell type were directly correlated to the amount of endogenous GCase RNA. Comparison of the GCase mRNA levels in Gaucher-versus non-Gaucher-derived cells revealed that in Gaucher cells this RNA is always more abundant than in the corresponding non-Gaucher counterparts, suggesting the involvement of a feed-back mechanism sensitive to the levels of actual enzymatic activity.

LIS1 and platelet-activating factor acetylhydrolase (Ib) catalytic subunits, expression in the mouse oocyte and zygote.

Platelet-activating factor is a phospholipid with several documented roles in the pre-implantation embryo. Enzymes that belong to the platelet-activating factor acetylhydrolases family inactivate platelet-activating factor. Cytosolic platelet-activating factor acetylhydrolase (Ib) is a heterotetramer composed of two catalytic subunits (alpha1/alpha2) and two regulatory LIS1 subunits. The expression of these components was monitored in the mouse oocytes and zygotes using reverse-transcribed PCR and Western blot analysis. Interestingly, these proteins are expressed in the oocyte and zygote and their expression increases after fertilization, probably due to stabilization of maternal RNA. Lis1 mRNA transcription also increases after fertilization. However, assaying for expression of a specific paternal LIS1 isoform detected no zygotic translation in the one cell stage. These findings suggest a potential role for platelet-activating factor acetylhydrolase (Ib) components in the early mouse embryo.

Human sphingolipid activator protein-1 and sphingolipid activator protein-2 are encoded by the same gene.

Mixed oligonucleotide primers complementary to the translation product of the sphingolipid activator protein (SAP)-2 were used to generate a 144-base pair (bp) complementary DNA (cDNA). This cDNA probe was used to isolate a 2,649-nucleotide-long cDNA that was sequenced and found to contain coding sequences for two known activators of lysosomal enzymes, namely, the sphingolipid activator protein (SAP)-1 and SAP-2. The cDNA contains an open reading frame of 1,482 nucleotides and 1,167 nucleotides of 3'-nontranslated region, followed by a stretch of 24 residues of adenylic acid. At 20 nucleotides upstream from the poly(A) tail there is a consensus AATAAA polyadenylation signal that is preceded by another potential polyadenylation signal. The cDNA, designed SAP-1/SAP-2 cDNA, hybridizes with two human mRNA species of approximately 3 kb in length, which most probably arise from polyadenylation at different sites. There are higher amounts of steady-state RNA levels of the SAP-1/SAP-2 mRNA in skin fibroblasts in comparison to B cells. The steady-state SAP-1/SAP-2 mRNA levels in Gaucher B cells are higher than in their normal counterparts. There is one human SAP-1/SAP-2 gene that has been cloned and is localized on two approximately 5 kb BamHI fragments.

Evidence for the involvement of the hippocampus in the pathophysiology of schizophrenia.

The hippocampus, a medial temporal lobe structure, is often considered to play an important role in the pathophysiology of schizophrenia. Recent developments of neuroimaging and molecular postmortem techniques have significantly increased our ability to study the role of discrete brain regions in the pathophysiology of schizophrenia. This article describes animal models, structural, histological, molecular biology, and neuropsychological evidence for the involvement of the hippocampus in the pathophysiology of schizophrenia. The major findings in schizophrenic patients are decreased volumes, hypometabolism, and cytoarchitectural abnormalities which are more robust on the left hippocampus, as well as verbal memory impairment. It is yet to be determined whether these changes are neurodevelopmental or neurodegenerative in nature. Overall, these findings indicate that there are subtle changes in the hippocampus of schizophrenic patients. More comprehensive and focused hippocampal research in schizophrenia is required to elucidate the contribution of this intriguing brain structure to the pathophysiology of schizophrenia.

The role of the electrogenic sodium pump in the potassium relaxation of the rabbit ear artery.

1. The mechanism of the potassium-induced relaxation of contracted vascular smooth muscle has been studied in helical strips of rabbit ear artery by recording isometric tension and membrane potential. The arteries were stimulated with a standard concentration of 2 x10(-8) M noradrenaline producing about 20% of the maximal contractile response to noradrenaline. 2. At K+ concentrations between 1.2 and 20.0 mM, a positive correlation was found between [K+]0 and the contractile response to noradrenaline, i.e. increasing [K+]0 enhanced the effect of noradrenaline. However, when noradrenaline was added after a 10 min exposure to reduced [K+]0 (1.2 mM) and [K+]0 was then increased to 5.9 mM after the tension had reached a plateau, a relaxation of about 50% occurred instead of the expected increase in tension. The relaxation was preceded by a membrane hyperpolarization of about 8 mV. 3. A biphasic change of both membrane potential and tension resulted when, after exposure of arterial strips to 1.2 mM K+ and stimulation with noradrenaline, [K+]0 was increased to 20.0 mM: hyperpolarization and relaxation developed faster than at 5.9 mM K+, but after 1 min the hyperpolarization changed to a depolarization followed by an increase in tension. 4. Ca2+-free solution and D 600 (an inhibitor of Ca2+ transmembrane flux) diminished, but did not abolish, the tension response to noradrenaline; in the presence of D 600, the hyperpolarization upon increasing [K+]0 after a previous exposure to 1.2 mM K+ was not affected, but a relaxation no longer occurred. 5. It is concluded that a) electrogenic ion transport causes the hyperpolarization that occurs when [K+]0 is increased after a period of exposure to low [K+]0; b) the hyperpolarization leads to relaxation by blocking the influx of extracellular Ca2+, but does not affect that component of the contractile response which is due to the release of intracellular Ca2+.

Action of D-600 on spontaneous and electrically stimulated activity of the parturient rat uterus.

The effects of D-600, the methoxy derivative of verapamil, on the parturient rat uterus have been studied. At concentrations of 10(-8) to 10(-7) M, D-600 abolishes spontaneous contractions and reduces the force of electrically evoked contractions. The first action is associated with a reduction in amplitude of the slow waves (oscillations of membrane potential which underlie rhythmic bursts of spikes initiating contractions). The second is associated with a reduction in frequency of spikes within the bursts accompanying the electrically stimulated contractions. This reduction results from a decrease in the slope of the prepotential in cells in pacemaker areas of the muscle. D-600 also decreased the height and rising velocity of the conducted action potentials, but these actions cannot account entirely for the reduction in contractile force.

Action of prostaglandin, PGF2alpha, on the uterus of the pregnant rat.

The effects of prostaglandin F2alpha (PG) have been studied on the transmembrane potentials and contractions in isolated myometrial strips from pregnant rats. The results showed that: 1. The sensitivity of the myometrium to exogenous PG increases from day 19 to day 22 of gestation. 2. The electrical response to PG, at maximally effective doses (10-6 to 10-5 M) consists of a slow depolarization which upon reaching threshold initiates spike discharge. 3. These actions are most pronounced at term (day 22) and are due to a direction of PG on the myometrial cells. 4. D-600 (a methoxy derivative of verapamil) abolishes spike discharge and the phasic contractions induced by PG but has no effect on the slow depolarization and the accompanying increase in tonic tension. 5. The slow depolarization is dependent upon the presence of sodium in the external environment and is unaffected by the removal of calcium. 6. The spikes (and phasic contractions) are dependent upon the presence of calcium in the external environment.

Abnormal cortical development; towards elucidation of the LIS1 gene product function (review).

Lissencephaly is a relatively common brain malformation. Lissencephaly type 1 is characterized by the smooth appearance of the cortex and the presence of four abnormally positioned layers instead of the normal six. Lissencephaly is considered to be an abnormality in neuronal migration. The gene mutated in type 1 lissencephaly was cloned by us and designated LIS1. Recently, several genes involved in cortical development have been cloned in the mouse. In human an additional X-linked lissencephaly gene has been identified. We summarize here our current knowledge on the LIS1 gene and its function. It has been identified as a non-catalytic subunit of PAF-acetylhydrolase, a heterotrimeric enzyme which inactivates the platelet-activating factor (PAF). In addition, we have demonstrated that LIS1 interacts with tubulin, and affects the dynamics properties of microtubles. LIS1 contains seven WD repeats and may structurally resemble the beta-subunit of heterotrimeric G proteins. Interestingly, the catalytic subunit of PAF-acetylhydrolase was found to resemble the alpha subunit of heterotrimeric G proteins. We raise the possibility that LIS1 is part of an intracellular signaling pathway involved in neuronal migration.

Missense mutations resulting in type 1 lissencephaly.

Proper human brain formation is dependent upon the integrated activity of multiple genes. Malfunctioning of key proteins results in brain developmental abnormalities. Mutation(s) in the LIS1 gene or the X-linked gene doublecortin (DCX) results in a spectrum of disorders including lissencephaly, or "smooth brain", and subcortical band heterotopia, or "doublecortex". Here, we will focus on a particular subset of missense mutations in these two genes and their effect on protein structure and function.

Expression of chLIS1, a chicken homolog of LIS1.

We have isolated the chicken LIS1 homolog, chLIS1, with DNA sequence similarity of over 68% to the human cDNA and 99% amino acid identity. Additionally, we describe the pattern of chLIS1 expression in the chicken embryo. The early embryonic expression is highly specific to the developing nervous system, whereas later the expression is more widespread.

Structural analysis of the human glucocerebrosidase genes.

Two different genomic clones containing the entire coding sequence of human glucocerebrosidase were isolated from a fetal liver library using a cDNA probe previously cloned by us. These clones correspond to two human glucocerebrosidase genes, designated 6-1 and 10-2. Clone 6-1 contains sequences homologous to the cDNA we cloned previously. The promoter regions of the genes were identified by S1 analysis and sequenced. They contain TATA- and CAAT-like boxes, but lack a GGCGGG motif. When coupled to the bacterial gene coding for chloramphenicol acetyl transferase (CAT) and transfected to Gaucher skin fibroblast lines, both promoter fragments enhanced CAT activity. The promoter of gene 6-1 was eight times more efficient than the promoter of gene 10-2. Northern blot analysis revealed three human glucocerebrosidase RNA species of 6, 2.6, and 2.2 kb in size. The 6-kb transcript is probably a nuclear transcript whereas the 2.6-kb and 2.2-kb transcripts are cytoplasmic species which emerge from polyadenylation at different sites.

Reduction of microtubule catastrophe events by LIS1, platelet-activating factor acetylhydrolase subunit.

Forming the structure of the human brain involves extensive neuronal migration, a process dependent on cytoskeletal rearrangement. Neuronal migration is believed to be disrupted in patients exhibiting the developmental brain malformation lissencephaly. Previous studies have shown that LIS1, the defective gene found in patients with lissencephaly, is a subunit of the platelet-activating factor acetylhydrolase. Our results indicated that LIS1 has an additional function. By interacting with tubulin it suppresses microtubule dynamics. We detected LIS1 interaction with microtubules by immunostaining and co-assembly. LIS1-tubulin interactions were assayed by co-immunoprecipitation and by surface plasmon resonance changes. Microtubule dynamic measurements in vitro indicated that physiological concentrations of LIS1 indeed reduced microtubule catastrophe events, thereby resulting in a net increase in the maximum length of the microtubules. Furthermore, the LIS1 protein concentration in the brain, measured by quantitative Western blots, is high and is approximately one-fifth of the concentration of brain tubulin. Our new findings show that LIS1 is a protein exhibiting several cellular interactions, and the interaction with the cytoskeleton may prove to be the mode of transducing a signal generated by platelet-activating factor. We postulate that the LIS1-cytoskeletal interaction is important for neuronal migration, a process that is defective in lissencephaly patients.