Endosome dynamics regulated by a Rho protein.

Vesicular transport is a dynamic process that requires coordinated interactions between membrane and cytoskeleton. The mechanisms and molecules integrating these interactions are unclear. A Rho protein, RhoD, might provide a molecular link between membrane traffic and the cytoskeleton. Activated RhoD causes rearrangements of the actin cytoskeleton and cell surface, and governs early endosome motility and distribution.

Co-operative regulation of endocytosis by three Rab5 isoforms.

Rab proteins are small GTPases involved in the regulation of membrane traffic. Rab5a has been shown to regulate transport in the early endocytic pathway. Here we report the isolation of cDNA clones encoding two highly related isoforms, Rab5b and Rab5c. The two proteins share with Rab5a all the structural features required for regulation of endocytosis. Rab5b and Rab5c colocalize with the both transferrin receptor and Rab5a, stimulate the homotypic fusion between early endosomes in vitro and increase the rate of endocytosis when overexpressed in vivo. These data demonstrate that three Rab5 isoforms cooperate in the regulation of endocytosis in eukaryotic cells.

Isolation of a murine cDNA clone encoding Rab19, a novel tissue-specific small GTPase.

Using a rapid amplification of cDNA ends (RACE) cloning approach, we have isolated a cDNA clone encoding Rab19, a novel small GTPase of the Rab subfamily contained within partial sequences previously described [Chavrier et al., Gene 112 (1992) 261-264]. Northern blot analysis of the distribution of the rab19 mRNA in various adult mouse tissues and NIH 3T3 fibroblasts revealed that rab19 is expressed in a tissue-specific manner. The rab19 transcript was detected at high levels in intestine, lung and spleen, and at a lower level in kidney. In contrast, liver, brain, heart and NIH 3T3 fibroblasts contain only very little or no detectable rab19 mRNA. Therefore, Rab19 is likely to represent a novel tissue- or cell type-specific small GTPase.

Cloning and subcellular localization of novel rab proteins reveals polarized and cell type-specific expression.

Small GTPases of the rab subfamily are specific regulators of vesicular transport. The intracellular localization of these proteins has been mostly investigated in cultured cells where they have been found associated with distinct compartments of the exocytic and endocytic pathways. Using a PCR-based cloning approach we have recently identified several novel rab proteins, extending the total number of this family to more than 30 members. Here, we have investigated the mRNA expression in different tissues and the intracellular localization in organ cryosections of two rab proteins, rab18 and rab20. Both northern blot analysis and confocal immunofluorescence microscopy demonstrated that these proteins are expressed in a tissue- and cell type-dependent manner. Despite their presence in non-polarized cells and polarized cells, both proteins are highly expressed on the apical side of kidney tubule epithelial cells. Electron microscopic studies revealed that rab18 and rab20 are located in apical dense tubules, endocytic structures underlying the apical plasma membrane, suggesting that they play a role in apical endocytosis/recycling. In intestinal epithelial cells as well, both proteins were localized apically, but, in addition, rab18 was found associated with the basolateral domain, suggesting that this protein is not restricted to the apical transport machinery of polarized epithelial cells. The results demonstrate that, depending on the epithelial cell type, rab proteins that are also expressed in non-polarized cells may be enriched in one or both surface domains. Together with the observed tissue- and cell type-dependent variation in the expression of the rab proteins, this suggests that the large number of mammalian rab proteins might reflect the specific requirements in the organization of membrane traffic encountered by different cell types.

Mapping of Ras-related GTP-binding proteins by GTP overlay following two-dimensional gel electrophoresis.

For identification of Rab, Rac, Rho, Ral, Rap, and Arf proteins on two-dimensional polyacrylamide gels, we have expressed full-length cDNAs of members of these protein families with the T7 RNA polymerase-recombinant vaccinia virus expression system. Membrane preparations from cells expressing the cDNAs were subjected to high-resolution two-dimensional polyacrylamide gel electrophoresis followed by [alpha-32P]GTP ligand blotting. We have mapped 28 small GTP-binding proteins relative to their isoelectric points and according to their molecular weights and by immunoblotting with specific antibodies. Rab and Rho proteins could be specifically identified by extraction of streptolysin O-permeabilized Madin-Darby canine kidney (MDCK) cells with Rab- and Rho-GDP dissociation inhibitor. We applied the reference mapping to analyze the GTP-binding patterns of synaptosome fractions from rat brain. The purified synaptosomes exhibited specific enrichment of Rab3a, Rab5a, Ral, and several other GTPases. This approach and the map we have produced should provide a useful aid for the analysis of the expression and localization of members of all families of small GTP-binding proteins in various cell types and subcellular fractions.

Rab5a is a common component of the apical and basolateral endocytic machinery in polarized epithelial cells.

In nonpolarized cells, the small GTPase Rab5a is localized to the plasma membrane, clathrin-coated vesicles, and early endosomes. Rab5a is required for early endosome fusion in vitro and regulates transport between the plasma membrane and early endosomes, in vivo. In polarized epithelial cells endocytosis occurs from separate apical and basolateral plasma membrane domains. Internalized molecules are initially delivered to distinct apical or basolateral early endosomes. In vitro, apical early endosomes can readily fuse with one another but not with the basolateral endosomes and vice versa, thereby indicating that the apical and basolateral early endocytic pathways are controlled by distinct machineries. Here, we have investigated the localization and function of Rab5a in polarized epithelial cells. Confocal immunofluorescence microscopy on mouse kidney sections revealed association of the protein with the apical and basolateral plasma membrane domains and underlying structures. In polarized Madin-Darby canine kidney I cells, endogenous and overexpressed Rab5a have the same distribution. Moreover, overexpression of the protein causes a 2-fold increase in fluid-phase uptake from both domains of the cell, thus showing that Rab5a functions in apical and basolateral endocytosis. Our data indicate that the apical and basolateral endocytic machineries of epithelial cells share common regulatory components and that Rab5a per se is not sufficient to target endocytic vesicles to apical or basolateral early endosomes.

Inhibition of rab5 GTPase activity stimulates membrane fusion in endocytosis.

Small GTPases of the rab family control distinct steps of intracellular transport. The function of their GTPase activity is not completely understood. To investigate the role of the nucleotide state of rab5 in the early endocytic pathway, the effects of two mutants with opposing biochemical properties were tested. The Q79L mutant of rab5, analogous with the activating Q61L mutant of p21-ras, was found to have a strongly decreased intrinsic GTPase activity and was, unlike wild-type rab5, found mainly in the GTP-bound form in vivo. Expression of this protein in BHK and HeLa cells led to a dramatic change in cell morphology, with the appearance of unusually large early endocytic structures, considerably larger than those formed upon overexpression of wild-type rab5. An increased rate of transferrin internalization was observed in these cells, whereas recycling was inhibited. Cytosol containing rab5 Q79L stimulated homotypic early endosome fusion in vitro, even though it contained only a small amount of the isoprenylated protein. A different mutant, rab5 S34N, was found, like the inhibitory p21-ras S17N mutant, to have a preferential affinity for GDP. Overexpression of rab5 S34N induced the accumulation of very small endocytic profile and inhibited transferrin endocytosis. This protein inhibited fusion between early endosomes in vitro. The opposite effects of the rab5 Q79L and S34N mutants suggest that rab5:GTP is required prior to membrane fusion, whereas GTP hydrolysis by rab5 occurs after membrane fusion and functions to inactivate the protein.

Laboratory diagnosis of radicular and pseudoradicular syndromes in cerebrospinal fluid (CSF): reliability of methods in consideration of pathogenetic aspects. Report on section "CSF diagnosis" of 4th Klagenfurter Neurology Workshop Conference on radicular and pseudoradicular syndromes, Klagenfurt, Austria, August 28-29, 1992.

Laboratory tests may be used to confirm the clinical differentiation of pseudoradicular syndromes and radicular syndromes. In the presence of pseudoradicular syndromes, CSF and blood samples yield no positive results with either non-specific or specific methods. Radicular syndromes give rise to positive findings; using non-specific methods they can be subdivided into inflammatory and non-inflammatory forms, with and without blood-nerve barrier impairment. Non-specific quantities of CSF routine diagnosis are total protein, albumin, leukocyte counts and differential cell count, L-lactate, intrathecal -IgG, -IgA, -IgM and immunoglobulin-class oligoclonal bands. Oligoclonal bands enable the highly sensitive differentiation of non-inflammatory from subacute-chronically inflammatory forms of radicular syndromes. Most of the specific quantities are the subject of current research, e.g. bacterial antigens, D-lactate, cultivation tests, polymerase chain reaction tests and pathogen-specific oligoclonal bands. Pathomechanisms affecting the permeability of the blood-nerve barrier to increasing concentrations of protein and to leukocyte subsets possibly explain the CSF findings in radicular and pseudoradicular syndromes.

Isolation of a mouse cDNA encoding Rab23, a small novel GTPase expressed predominantly in the brain.

The full-length cDNA encoding Rab23, a novel Ras-related small GTPase, was isolated using the sequence of a previously described [Chavrier et al., Gene 112 (1992) 261-264] short cDNA fragment and the rapid amplification of cDNA ends (RACE) PCR techniques. The deduced amino acid sequence was not very closely related to any previously described small GTPase, but was within the Rab subfamily. A Northern analysis revealed that the rab23 mRNA is predominantly expressed in the brain, which places the protein, together with Rab3a and Rab15, in the group of small GTPases characteristic of the nervous system.

Molecular cloning and subcellular localization of three GTP-binding proteins of the rab subfamily.

Small GTPases of the rab subfamily are involved in regulation of intracellular membrane transport events. We recently used a PCR approach to isolate short cDNA fragments of a number of novel rab sequences. These PCR fragments have not been used with cDNA library screening and PCR-based techniques to clone the cDNAs encoding three of these proteins, rab12, rab22, and rab24. By northern blot analysis, the messages were found to be present in a wide variety of mouse tissues. However, quantitative differences in the mRNA levels between the tissues were detected. We determined the subcellular localization of the GTPases by expressing the c-myc epitope-tagged proteins with the Semliki Forest virus and the vaccinia T7 vector systems. Transiently expressed rab12 was localized to the Golgi complex. This localization was confirmed using a polyclonal anti-peptide antibody detecting the endogenous protein in BHK cells. rab22 expressed from the cDNA was localized to endosomal compartments and to the plasma membrane. After longer periods of expression, the protein was found on abnormally large perinuclear endosomal structures, suggesting that it is a potent regulator of events in the endocytic pathway. Finally, rab24 was found in the endoplasmic reticulum/cis-Golgi region and on late endosomal structures. The localization of rab24 may indicate its involvement in autophagy-related processes.

Transport and production of cerebrospinal fluid (CSF) change in aging humans under normal and diseased conditions.

In three control groups of lumbar (L), cisternal (C), and ventricular (V) cerebrospinal fluid (CSF) with prealbumin contents of ventricular origin (CSF-V-prealbumin) within the reference range, indications for an increased transport of V-CSF into spinal space are presented in the elderly as well as an elevated production of V-CSF. Indications for a diminished V-CSF production, respectively, an increased transport of CSF-V-prealbumin into the spinal space were found in elderly patients suffering from diverse CNS diseases. As these alterations proved to be not disease-specific, the age-related increase of CSF transport was discussed with respect to brain atrophy and enlarged CSF spaces in the elderly central nervous system (CNS). CSF serum prealbumin content, an indicator of blood-brain-barrier function, increased with age similarly to albumin in the "normal" elderly, respectively with diverse CNS diseases. Age- and disease-related decreases of albumin and prealbumin contents in blood serum may lead to wrong interpretations.

Rab17, a novel small GTPase, is specific for epithelial cells and is induced during cell polarization.

The rab subfamily of small GTPases has been demonstrated to play an important role in the regulation of membrane traffic in eukaryotic cells. Compared with nonpolarized cells, epithelial cells have distinct apical and basolateral transport pathways which need to be separately regulated. This raises the question whether epithelial cells require specific rab proteins. However, all rab proteins identified so far were found to be equally expressed in polarized and nonpolarized cells. Here we report the identification of rab17, the first epithelial cell-specific small GTPase. Northern blot analysis on various mouse organs, revealed that the rab17 mRNA is present in kidney, liver, and intestine but not in organs lacking epithelial cells nor in fibroblasts. To determine whether rab17 is specific for epithelial cells we studied its expression in the developing kidney. We found that rab17 is absent from the mesenchymal precursors but is induced upon their differentiation into epithelial cells. In situ hybridization studies on the embryonic kidney and intestine revealed that rab17 is restricted to epithelial cells. By immunofluorescence and immunoelectron microscopy on kidney sections, rab17 was localized to the basolateral plasma membrane and to apical tubules. Rab proteins associated with two distinct compartments have been found to regulate transport between them. Therefore, our data suggest that rab17 might be involved in transcellular transport.

[Electroencephalography and cranial computed tomography in a posterior infarct]. / Elektroenzephalographie und kranielle Computertomographie beim Posteriorinfarkt.

In this study we interpreted the electroencephalograms of 50 patients, all suffering from a cerebral infarction occurred in the circulation area of A. cerebri posterior with or without involvement of thalamic structures. The diagnosis was established by clinical findings and via CT-examinations. Besides the EEG-findings which always occurred in the occipital region we found alterations which exceeded the above mentioned posterior disorder of the brain function. In most cases these have been bilateral paroxysmal disorders projected into the temporal regions but lateralized to the region of the existing posterior infarction. If we had CT-examinations of cerebral infarctions in the circulation area of A. cerebri posterior without an involvement of thalamic structures, there was no projected activity which exceeded the posterior disorder in EEG. In all cases with the additional affection of thalamic structures in CT the bilateral paroxysmal EEG disorders into the temporal region were found.

The importance of MRI (magnetic resonance imaging) for the diagnosis of brainstem infarction.

As experience with it accumulates, high resolution MRI seems more capable of representing morphological alterations of the central nervous system than cranial computer tomography (CCT). The question thus arises whether the detection of brainstem infarctions with MRI might not be easier than with CCT and how the topography of brainstem lesions demonstrated with MRI correlate with clinical symptoms and signs. 16 patients with definite clinical symptoms of brainstem infarction were examined by MRI. The examination took place between the 10th and 14th day after the occurrence of the infarction. Using MRI, we found pathological results in only 8 of these cases. In only 5 of these cases a clear relationship between the neurological picture and the topography of the lesion could be shown. These findings demonstrate that despite the high quality of the MRI images, the clinical examination for symptoms and signs is more important than imaging in the brainstem infarction.

[Idiopathic hydrocephalus in adolescence]. / Idiopathischer Hydrozephalus in der Adoleszenz.

BACKGROUND: Decompensation of chronic idiopathic hydrocephalus can occur at every age, but seems to be rather frequent in the middle of the second decade. From this observation, the question arises, whether or not in these cases a special manifestation of hydrocephalus occurs and, should the situation arise, whether this finding might influence the discussion about pathogenesis. METHODS: We give some casuistic material about six hydrocephalic patients who became symptomatic between their 12th and 16th year of life. RESULTS: All of these patients showed a tri-ventricular appearance of hydrocephalus as well as radiological signs of chronicly elevated intracranial pressure, while clinical signs and symptoms differed considerably. These findings are linked to the discussion of the pathogenesis of so-called aqueductal stenosis. CONCLUSIONS: Whenever, after normal development in childhood, symptoms like increasing headache, poor concentration, dizziness or disturbances of gait appear during puberty, X-ray diagnosis of the skull is recommended. If it shows the signs of chronic elevated intracranial pressure, further investigations are necessary.

[Epithelial cyst of the central nervous system. A rare abnormality]. / Die epitheliale Zyste des ZNS. Eine seltene Fehlbildung.

A cystic process in the right frontal lobe of the brain in a man of 35 years of age, and an intramedullary cyst in the upper cervical spinal cord in a 29-year old woman, are described. In both cases, the wall of the cyst consisted of mucosal cells and cells having cilia pointing to the inside, located on a thin tissue layer, the structure of which resembled that of a meningioma. Central nervous cysts of this kind are usefully classified together with other, similar cysts (bronchogenic, ependymal and others) as "epithelial cysts". They must be differentiated from neoplastic tumours. Analysis of the cystic contents can prove helpful.

[Central pontine myelinolysis following severe hyponatremia]. / Zentrale pontine Myelinolyse nach ausgeprägter Hyponatriämie.

Central pontine myelinolysis is a process of demyelinisation with variable neurological symptoms related to the localization. Predisposing factors are alcoholism and malnutrition. Rapid correction of severe hyponatremia is suspected to be a primary cause for central pontine myelinolysis. We report a 43 year old chronic alcoholic and polytoxicomanic female patient, who was admitted comatose with a serum sodium level of 94 mmol/l, caused by a syndrome of inappropriate ADH secretion. After initial improvement under careful sodium correction, the patients neurologic condition degraded progressively and within 4 weeks she developed a "locked-in"-syndrome. Only then the suspected central pontine myelinolysis could be demonstrated in nuclear magnetic resonance and computer tomography. We presume that, although sodium correction was done relatively slowly in this patient, it probably contributed to her development of central pontine myelinolysis all the same. Due to this case we review the literature on correction of hyponatremia, which shows growing evidence that it should start early but be continued very slowly (rise in serum-Na: max. 0.6 mmol/l/h) and requires frequent laboratory controls.

EEG development in early treated PKU patients from birth to 6 years of age.

In 126 early treated PKU patients (type I and type II) a close EEG follow up was performed from birth up to 6 years of age. A total of 1465 EEGs were performed before and after onset of dietary treatment and on 11 more subsequent occasions. The composition of the background activity was normal up to 6 years when only a small number of the children (19) showed no dominant alpha activity. The frequency of epileptiform activity of generalised as well as focal type was low in the first 2 years of life, but afterwards slightly enhanced in comparison to normal control groups. Other findings like generalised theta paroxysms or focal slow waves were rarely observed. Under a standardised protein load at 6 months (52 patients) and at 5 years of age (42 patients) a moderate generalised slowing of the background activity but no other abnormalities were noted.

Dumb-bell ganglioneuroma of the spine misinterpreted as progressive idiopathic scoliosis. Case report.

A giant ganglioneuroma generating a progressive scoliosis in a 16-year-old girl is presented. The interval between the start of the orthopaedic treatment and the diagnosis of the true nature of the disease was more than 4 years, thus allowing the development of a giant partly intracanalicularly partly retroperitonealy expanding tumor mass. The report emphasizes and describes the combined neurosurgical, general surgical and orthopaedic surgical treatment and presents the results of light- and electron microsopical, immunohistochemical and quantitative neurochemical investigations of the resected tumor.