Targeting myelin lipid metabolism as a potential therapeutic strategy in a model of CMT1A neuropathy.

In patients with Charcot-Marie-Tooth disease 1A (CMT1A), peripheral nerves display aberrant myelination during postnatal development, followed by slowly progressive demyelination and axonal loss during adult life. Here, we show that myelinating Schwann cells in a rat model of CMT1A exhibit a developmental defect that includes reduced transcription of genes required for myelin lipid biosynthesis. Consequently, lipid incorporation into myelin is reduced, leading to an overall distorted stoichiometry of myelin proteins and lipids with ultrastructural changes of the myelin sheath. Substitution of phosphatidylcholine and phosphatidylethanolamine in the diet is sufficient to overcome the myelination deficit of affected Schwann cells in vivo. This treatment rescues the number of myelinated axons in the peripheral nerves of the CMT rats and leads to a marked amelioration of neuropathic symptoms. We propose that lipid supplementation is an easily translatable potential therapeutic approach in CMT1A and possibly other dysmyelinating neuropathies.

Hypoxia-stimulated membrane trafficking requires T-plastin.

AIM: Traffic between the plasma membrane and the endomembrane compartments is an essential feature of eukaryotic cells. The secretory pathway sends cargoes from biosynthetic compartments to the plasma membrane. This is counterbalanced by a retrograde endocytic route and is essential for cell homoeostasis. Cells need to adapt rapidly to environmental challenges such as the reduction of pO2 which, however, has not been analysed in relation to membrane trafficking in detail. Therefore, we determined changes in the plasma membrane trafficking in normoxia, hypoxia, and after reoxygenation. METHODS: Membrane trafficking was analysed by using the bulk membrane endocytosis marker FM 1-43, the newly developed membrane probe mCLING, wheat germ agglutinin as well as fluorescently labelled cholera toxin subunit B. Additionally, the uptake of specific membrane proteins was determined. In parallel, a non-biased SILAC screen was performed to analyse the abundance of membrane proteins in normoxia and hypoxia. RESULTS: Membrane trafficking was increased in hypoxia and quickly reversed upon reoxygenation. This effect was independent of the hypoxia-inducible factor (HIF) system. Using SILAC technology, we identified that the actin-bundling protein T-plastin is recruited to the plasma membrane in hypoxia. By the use of T-plastin knockdown cells, we could show that T-plastin mediates the hypoxia-induced membrane trafficking, which was associated with an increased actin density in the cells as determined by electron microscopy. CONCLUSION: Membrane trafficking is highly dynamic upon hypoxia. This phenotype is quickly reversible upon reoxygenation, which suggests that this mechanism participates in the cellular adaptation to hypoxia.

Myelinated mouse nerves studied by X-ray phase contrast zoom tomography.

We have used X-ray phase contrast tomography to resolve the structure of uncut, entire myelinated optic, saphenous and sciatic mouse nerves. Intrinsic electron density contrast suffices to identify axonal structures. Specific myelin labeling by an osmium tetroxide stain enables distinction between axon and surrounding myelin sheath. Utilization of spherical wave illumination enables zooming capabilities which enable imaging of entire sciatic internodes as well as identification of sub-structures such as nodes of Ranvier and Schmidt-Lanterman incisures.

Concentration of rafts in platelet filopodia correlates with recruitment of c-Src and CD63 to these domains.

The molecular mechanism that causes non-adhesive, discoid platelets to transform into sticky dendritic bodies that form blood clumps is a complex series of events. Recently it has become clear that lipid microdomains--also known as rafts--play a crucial role in this process. We have used a non-cytolytic derivative of perfringolysin-O, a cholesterol binding cytolysin, that binds selectively to cholesterol-rich membrane domains, combined with confocal- and immunoelectron microscopy to visualize cholesterol-raft dynamics during platelet adhesion. In resting platelets cholesterol was uniformly distributed on the cell surface and confined to distinct intracellular compartments (i.e. multivesicular bodies, dense granules, and the internal membranes of alpha-granules). Upon interaction with fibrinogen, cholesterol accumulated at the tips of filopodia and at the leading edge of spreading cells. Stimulation with thrombin receptor activating peptide (TRAP) resulted in a similar redistribution of cholesterol towards filopodia. The adhesion-dependent raft aggregation was accompanied by concentration of the tyrosine kinase c-Src and the tetraspanin CD63 in these domains, whereas glycoprotein Ib (GPIb) was not selectively targeted to the raft clusters. c-Src, the tetraspanin CD63, and GPIb were recovered in biochemically isolated low-density membrane fractions. Disruption of rafts by depleting membrane cholesterol had no effect on platelet shape change but inhibited platelet spreading on fibrinogen and TRAP-induced aggregation. Our results demonstrate that cholesterol rafts in platelets are dynamic entities in the membrane that co-cluster with the tyrosine kinase c-Src and the costimulatory molecule CD63 in specialized domains at the cell surface, thereby providing a possible mechanism in functioning as signaling centres.

Recycling compartments and the internal vesicles of multivesicular bodies harbor most of the cholesterol found in the endocytic pathway.

We employed our recently developed immuno-electron microscopic method (W. Möbius, Y. Ohno-Iwashita, E. G. van Donselaar, V. M. Oorschot, Y. Shimada, T. Fujimoto, H. F. Heijnen, H. J. Geuze and J. W. Slot, J Histochem Cytochem 2002; 50: 43-55) to analyze the distribution of cholesterol in the endocytic pathway of human B lymphocytes. We could distinguish 6 categories of endocytic compartments on the basis of morphology, BSA gold uptake kinetics and organelle marker analysis. Of all cholesterol detected in the endocytic pathway, we found 20% in the recycling tubulo-vesicles and 63% present in two types of multivesicular bodies. In the multivesicular bodies, most of the cholesterol was contained in the internal membrane vesicles, the precursors of exosomes secreted by B cells. Cholesterol was almost absent from lysosomes, that contained the bulk of the lipid bis(monoacylglycero)phosphate, also termed lysobisphosphatidic acid. Thus, cholesterol displays a highly differential distribution in the various membrane domains of the endocytic pathway.

Angiotropic large-cell lymphoma presenting as pulmonary small vessel occlusive disease.

Angiotropic lymphoma (AL) is an unusual variant of extranodal lymphoma, characterized by massive proliferation of neoplastic lymphoid cells almost exclusively within blood vessels. Whereas the lymphoid origin of this disease is widely accepted it still remains unclear whether AL is a distinct entity that originates in the blood vessels or whether it represents a form of secondary intravascular dissemination of a primary solid lymphoma. The present case is unusual because death by right heart failure owing to extensive intravascular proliferation of neoplastic cells and subsequent occlusion of pulmonary blood vessels has not been described so far. In addition, the patient had suffered from a solid deposit of a large-cell B-lymphoma months before the angiotropic manifestation, suggesting that AL might develop out of more common types of non-Hodgkin's lymphomas.

Gangliosides are transported from the plasma membrane to intralysosomal membranes as revealed by immuno-electron microscopy.

A biotin-labeled derivative of the ganglioside GM1 (biotin-GM1) was used to study its transport along the endocytic pathway of cultured fibroblasts by immuno-electron microscopy. Using electron dense endocytic tracers we could demonstrate that late endosomes and lysosomes of these cells are long living organelles with a high content of internal membranes. Our studies show that during endocytosis the biotin-GM1 was transported to these intraendosomal and intralysosomal membranes. These observations support the hypothesis that glycosphingolipids (GSL) are preferentially degraded in intralysosomal vesicles.

Intracellular distribution of a biotin-labeled ganglioside, GM1, by immunoelectron microscopy after endocytosis in fibroblasts.

A radioactive and biotin-labeled analogue of GM1 (biotin-GM1) was synthesized which enabled us to analyze its intracellular distribution in the compartments of the endocytic route by electron microscopic immunocytochemistry using thin sections of human skin fibroblasts labeled with gold-conjugated antibiotin antibodies. Metabolic studies with the biotin-GM1 showed its partial degradation to the corresponding GM2 and GM3 derivatives. Further degradation was inhibited by the biotin residue. The distribution of biotin-GM1 after uptake by cells was studied by postembedding labeling techniques. On the plasma membrane the biotin-GM1 was detectable in the form of patches (0.1 micrometer in diameter), in caveola-like structures and, to a much lesser extent, in coated pits or vesicles. During endocytic uptake, the biotin-GM1 became detectable in organelles identified as late endosomes and lysosomes. The intracellular distribution of the biotin-GM1 was compared to the localization of the EGF receptor in EGF-stimulated fibroblasts. Both the biotin-GM1 and the EGF receptor were transported to intraendosomal and intralysosomal membranes, indicating that both membrane constituents follow the same pathway of endocytosis. Our observations show that biotin-GM1 can be successfully incorporated into the plasma membrane and be used as a tool for morphological detection of its pathway to lysosomes.

Accumulation of sphingolipids in SAP-precursor (prosaposin)-deficient fibroblasts occurs as intralysosomal membrane structures and can be completely reversed by treatment with human SAP-precursor.

The degradation of glycosphingolipids takes place in lysosomes by action of specific exohydrolases, with the assistance of sphingolipid activator proteins (SAPs). Four of the SAPs, SAP-A to -D (also called saposins A to D), are synthesized from a single protein, the SAP-precursor (prosaposin). Deficiency in this precursor protein, a rare inherited disease in humans, results in the storage of sphingolipids with short oligosaccharide head groups within the patients' tissues, and electron microscopy revealed the accumulation of large multivesicular storage organelles. In this study we analyze the multivesicular storage organelles in cultivated fibroblasts from these patients. The results support our hypothesis that endocytosis of plasma membrane-derived lipids occurs via small intraendosomal and intralysosomal vesicles and membrane structures that are then digested within the lysosomes (Sandhoff, K., T. Kolter, Trends in Cell Biol. 6, 98-103 (1996). First, we show that the storage compartment consists of late endosomes and lysosomes by immunogold labeling for marker proteins of these organelles. The transport of endocytosed bovine serum albumin-colloidal gold or cationized ferritin into the compartment occurs with the timing expected for transport to late endocytic organelles. Second, complementation of the medium of the SAP-precursor-deficient fibroblasts with only nanomolar concentrations of purified SAP-precursor nearly completely reversed the aberrant accumulation of multivesicular structures, thereby abolishing most of the intralysosomal membrane structures. Analysis of the sphingolipid pattern of the cells after metabolic labeling with [14C]serine reveals that the cells' ability to degrade glycosphingolipids is completely restored by feeding of SAP-precursor at the same concentrations. This is the first demonstration in vivo that endocytosed SAP-precursor is processed into functional active SAPs A,- B,- C, and D and that the degradation of the vesicular structures within the lysosomes depends on the presence of the SAPs. Moreover, these studies suggest that a therapy program based on feeding purified SAP-precursor may be valuable in treating the entire family of diseases which result from the loss of one or more of the SAPs.

Progressive liver failure in a patient with adult Niemann-Pick disease associated with generalized AL amyloidosis.

We report a case in which an adult form of Niemann-Pick disease (type B of NPD) was associated with a rapidly progressive generalized AL amyloidosis of kappa type. Both diagnosis were made by biopsy, the NPD by bone marrow biopsy and fibroblast culture, the amyloidosis by liver biopsy. Malignant non-Hodgkin lymphoma was not found. The patient, a 67-year-old woman, died from hepatic coma subsequent to a progressive liver failure. We discuss possible relations between the lysosomal storage disease and the development and rapid progression of amyloidosis.

[Pass-through operation for surgical treatment of recto-vaginal fistula (author's transl)]. / Die operative Therapie der Rektum-Scheiden-Fistel mit Hilfe der "Durchziehfistelplastik".

Recto-vaginal fistula is comparatively rare. There are certain cases in which it can be removed by means of the pass-through operation, a technique which bears good promise, with no need for cutting through the perineum into the rectum.

[The maternal and perinatal mortality and morbitity in attendance to Caesarean section in the period from 1956--1976 at the UFK-Jena (author's transl)]. / Die mütterliche und kindliche Mortalität und Morbidität nach Sectio im Zeitraum von 1956--1976 an der Universitäts-Frauenklinik Jena.

In the period from 1956--76 1 468 caesarean sections were accomplished at the UFK-Jena. 44 043 children were delivery during the 21-year period. The number of caesarean sections rose from 1,08% to 1956 to 10,8% in 1976. The fetal indication in 1956 was zero, on the other side it amounts to 34,3% in 1976. First place of the maternal indication in the 21-year period took in the cephalo pelvine disproportion. In the same time the maternal mortality in attendance of caesarean section was 12,3%. Considering the period between 1969--76, the maternal mortality fell to 2%. Maternal morbidity diminished from 1965 and obtained 23% in the period between 1974/1976. The feverish childbed took in the first place of the maternal morbidity (30%). The perinatal mortality fall from 28,57% in the period between 1956/1958 to 3,71% in the period between 1974/1976. The perinatal morbidity we could'nt state certainly. In 8,3% we found perinatal morbidity by acidosis. 5,5% of children, delivered by caesarean section needed medical care. 16,4% of all children were either small for gestational age or immature. The problem of caesarean section as an important method for termination of labor is discussed.

[Connatal leukaemia and pseudoleukaemia--two cases with cytochemical findings (author's transl)]. / Konnatale Leukämie und Pseudoleukämie--Zwei Fälle mit zytochemischen Befunden.

Two newborn infants with hyperleukocytosis and blast cells in the peripheral blood are compared. Case 1 represents a connatal leukemia, which could be characterized as monocytic by the naphthol-ASesterase reaction, which was inhibited by NaF. The child died after a few hours. Case 2 must be diagnosed as "pseudoleukemia" in mongolism. The blast cells were classified as lymphoblastic on the basis of a strong granular beta-glucuronidase reaction. The patient achieved spontaneous remission.

[Perinatal mortality and morbidity for childbirths from breech presentation at Gynaecological Hospital of Jena University, between 1966 and 1976/(77) (author's transl)]. / Die perinatale Mortalität und Morbidität bei Geburten aus Beckenendlage im Zeitraum von 1966 bis 1976/77 an der Universitäts-Frauenklinik Jena.

Studies were conducted at the Gynaecological Hospital of Jena University, between 1966 and 1976, into perinatal mortality and morbidity, in the context of 950 childbirths from breech presentation. Unambiguous decline in uncleared perinatal mortality was characterised by a numerical drop from 11.7. per cent in 1966/67 to 6.3 per cent in 1976/77.-Cleared perinatal mortality went down, similarly, for the same period of time, from 6.1 per cent to 2.5 per cent.-Uncleared perinatal mortality in the context of caesarean section was 0.65 per cent, while that related to vaginal delivery amounted to ten per cent. The cleared figures (after correction) were 0.54 and 5.4 per cent, the difference having been statistically secured with high significance. A decisive role was played, in that context, by the introduction of selective section as of 1975. Intranatal mortality has ever since dropped to zero, as compared to 1.9 per cent between 1966 and 1974.-Acidosis morbidity due to breech presentation was three times as high as that associated with cephalic delivery. The Apgar score was zero in 6.4 per cent of all children born by vaginal delivery. Therapeutic consequences are discussed.

[Surgical obstetrics in past and presence (author's transl)]. / Die operative Geburtshilfe in Vergangenheit und Gegenwart.

The history of surgical obstetrics is highlighted by discussing the historical development of forceps delivery, vacuum extraction, forceps vectis (application of a single blade only), embryotomy, and caesarean section operations. Finally present status of operative obstetrics is outlined from a modern point of view.