Neuroprotection by a caspase inhibitor in acute bacterial meningitis.

Half of the survivors of bacterial meningitis experience motor deficits, seizures, hearing loss or cognitive impairment, despite adequate bacterial killing by antibiotics. We demonstrate that the broad-spectrum caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl-ketone (z-VAD-fmk) prevented hippocampal neuronal cell death and white blood cell influx into the cerebrospinal fluid compartment in experimental pneumococcal meningitis. Hippocampal neuronal death was due to apoptosis derived from the inflammatory response in the cerebrospinal fluid. Apoptosis was induced in vitro in human neurons by inflamed cerebrospinal fluid and was blocked by z-VAD-fmk. As apoptosis drives neuronal loss in pneumococcal meningitis, caspase inhibitors might provide a new therapeutic option directed specifically at reducing brain damage.

Neurothelin: an inducible cell surface glycoprotein of blood-brain barrier-specific endothelial cells and distinct neurons.

The blood-brain barrier is characterized by still poorly understood barrier and transport functions performed by specialized endothelial cells. Hybridoma technology has been used to identify a protein termed neurothelin that is specific for these endothelial cells. Neurothelin is defined by the species-specific mouse mAb 1W5 raised against lentil-lectin-binding proteins of neural tissue from embryonic chick. In the posthatch chick, neurothelin expression is found on endothelial cells within the brain but not on those of the systemic vascular system. Injection of the monoclonal antibody in vivo leads to labeling of brain capillaries, indicating that the corresponding antigen is expressed on the luminal surface of brain endothelial cells. Transplantation of embryonic mouse brain onto the chick chorioallantoic membrane results in rodent brain vascularization by the avian vascular system. Subsequently, normally mAb 1W5-negative endothelial cells, originating from blood vessels of the chick chorioallantoic membrane, are induced to express neurothelin when they are in contact with mouse neural tissue. In contrast to differentiated brain neurons that do not express neurothelin, neurons of the nonvascularized chick retina synthesize neurothelin. However, neurothelin is not found on retinal ganglion cell axons terminating on 1W5-negative brain cells. 1W5 immunoreactivity was also found in the pigment epithelium that forms the blood-eye barrier. Putting epithelial cells into culture results in concentration of neurothelin at cell-cell contact sites, leaving other cell surface areas devoid of antigen. Therefore, the distribution of neurothelin appears to be regulated by cell-cell interactions. In Western blot analysis, neurothelin was identified as a protein with a molecular mass of approximately 43 kD. The protein bears at least one intramolecular disulfide bridge and sulfated glucuronic acid as well as alpha-D-substituted mannose/glucose moieties. The exclusive neurothelin expression in the posthatch chick on endothelial cells of the central nervous system but not on systemic endothelial cells makes neurothelin a marker specific for blood-brain barrier-forming endothelial cells. The spatiotemporally regulated neurothelin expression in neurons suggests an interaction between vascularization and neuronal differentiation.

Requirement for Atm in ionizing radiation-induced cell death in the developing central nervous system.

Ataxia telangiectasia (AT) is characterized by progressive neurodegeneration that results from mutation of the ATM gene. However, neither the normal function of ATM in the nervous system nor the biological basis of the degeneration in AT is known. Resistance to apoptosis in the developing central nervous system (CNS) of Atm-/- mice was observed after ionizing radiation. This lack of death occurred in diverse regions of the CNS, including the cerebellum, which is markedly affected in AT. In wild-type, but not Atm-/- mice, up-regulation of p53 coincided with cell death, suggesting that Atm-dependent apoptosis in the CNS is mediated by p53. Further, p53 null mice showed a similar lack of radiation-induced cell death in the developing nervous system. Atm may function at a developmental survival checkpoint that serves to eliminate neurons with excessive DNA damage.

c-jun Is dispensable for developmental cell death and axogenesis in the retina.

Although a number of studies have implicated c-Jun in neuronal death and axonal regeneration, it is unknown whether Jun function is essential for either response. One approach to resolve this issue is to analyze knock-out mice. However, c-jun-null mice die at midgestation, precluding critical investigation. Therefore, a xenograft paradigm was used in which retinas from embryonic day 12.5 (E12.5) c-jun nullizygous or wild-type mice were transplanted onto the superior colliculus of newborn rats. The rats were allowed to develop, and the grafts were assayed at various times for cell death and axon growth. Histologically, grafts of both genotypes developed in identical manners and had morphological characteristics of retinas. A functional c-jun allele was not essential for axogenesis, because ganglion cells in retinal grafts from c-jun nullizygous mice developed axons that projected into the colliculus. Programmed cell death (PCD) was also evident in the age-appropriate regions of the retina in both wild-type and c-jun-null grafts. Furthermore, there were no discernible differences in the number or location of dying cells in the two genotypes. That c-jun was not essential for PCD was supported by two additional findings. First, a c-jun-lacZ reporter gene was expressed in many cells in developing and grafted retinas, although only a few of these cells were destined to die. Second, in E12.5 c-jun-null embryos there were normal levels of PCD in the trigeminal ganglion. Together, these data indicate that c-Jun is not essential for axon growth in the retina or for PCD in the retina and trigeminal ganglion.

The Ca2+ binding protein, frequenin is a nervous system-specific protein in mouse preferentially localized in neurites.

Frequenin is a Ca2+-binding protein that has been implicated in the regulation of neurotransmitter release at the neuromuscular junction [15,16]. However, its cellular and subcellular localization in brain have not been determined. Therefore, we cloned mouse frequenin (Mfreq) and investigated its expression both in vivo and in vitro. The amino acid sequence of Mfreq is homologous to that of frequenins from other species. Northern and Western blot analyses indicated that the Mfreq mRNA is a single species of 4.2 kb, and that the protein has a mass of 24 kDa protein on SDS gel, respectively. Expression of Mfreq is nervous system specific. However, Mfreq mRNA and protein are widely distributed in the brain, spinal cord, and dorsal root ganglia. Mfreq is expressed in early embryonic brain and the levels of Mfreq remain high throughout development. In situ hybridization and immunocytochemistry demonstrated that Mfreq is expressed primarily in neurons and presumptive astrocytes. The Mfreq protein was preferentially localized in neurites (dendrites and axons). Double immunofluorescence microscopy established that Mfreq was co-localized with the dendritic marker, MAP-2 and the synapse marker, SV2 in cultured hippocampal neurons. The distribution and subcellular localization of Mfreq may help understand its cellular function.

[Esophagus carcinoid--a rare differential diagnosis in malignant esophageal neoplasia]. / Das Oesophaguscarcinoid--eine seltene Differentialdiagnose maligner Oesophagusneoplasien.

Two of patients with a carcinoid tumor of the esophagus are reported, an extremely rare localisation. Eight months after esophagectomy the 50-year-old male is entirely well and without any sign of recurrence; the 56-year-old female has died in this time. Our two cases are discussed in comparison with seven cases found in the literature.

Ultramicroscopic investigations of the exocrine pancreas of the dog after vagotomy.

The exocrine pancreas of 20 dogs was investigated electron microscopically before and 3, 4, 10, 11 and 14 days after bilateral truncular vagotomy. Up to the tenth day of the experiment a disturbance of the secretion of the acinar cells could be found with a sustained increase in enzyme protein synthesizing and lysosomal activity, irregular formation of zymogen granules and probably continuous extrusion of the enzyme proteins. The possible regulation mechanisms after vagotomy are discussed.

[Ultrasound differential diagnostic aspects in cystadenoma of the pancreas]. / Sonographisch-differentialdiagnostische Aspekte beim Zystadenom des Pankreas.

AIM: Assessment of differential diagnostic criteria of cystic adenomas of the pancreas. METHOD: We rechecked on all diagnostic criteria of patients with pancreatic pseudocysts retrospectively who had been treated in our department between 1981 and 1993. RESULTS: 12 patients with cystic adenomas of the pancreas had been treated in our department i.a. 7.8% of all cystic pancreatic tumours. Histopathologically 1 microcystic and 4 macrocystic adenomas as well as 7 cystic adenocarcinomas were seen. CONCLUSION: Diagnostic ultrasound criteria are discussed. Ultrasound-guided fine--needle-biopsy (FNB) is necessary to diagnose the content as well as the status of the pancreatic cysts. The ultrasound and the fine-needle-biopsy findings are important for differentiating between cystic adenomas and pseudocysts of the pancreas.

Expression of the BDNF gene in the developing visual system of the chick.

Using a sensitive and quantitative method, the mRNA levels of brain-derived neurotrophic factor (BDNF) were determined during the development of the chick visual system. Low copy numbers were detected, and BDNF was found to be expressed in the optic tectum already 2 days before the arrival of the first retinal ganglion cell axons, suggesting an early role of BDNF in tectal development. After the beginning of tectal innervation, BDNF mRNA levels markedly increased, and optic stalk transection at day 4 (which prevents subsequent tectal innervation) was found to reduce the contralateral tectal levels of BDNF mRNA. Comparable reductions were obtained after injection of tetrodotoxin into one eye, indicating that, already during the earliest stages of target encounter in the CNS, the degree of BDNF gene expression is influenced by activity-dependent mechanisms. BDNF mRNA was also detected in the retina itself and at levels comparable to those found in the tectum. Together with previous findings indicating that BDNF prevents the death of cultured chick retinal ganglion cells, these results support the idea that the tightly controlled expression of the BDNF gene might be important in the co-ordinated development of the visual system.

[Determination of the isoenzymes of alkaline phosphatase in serum and tissue homogenates with HPLC]. / Bestimmung der Isoenzyme der alkalischen Phosphatase im Serum und in Gewebehomogenaten mit HPLC.

We describe a new method for separating alkaline phosphatase isoenzymes by "high performance" liquid chromatography. Isoenzymes are chromatographed on the column (Mono Q HR 5/5, a strong anion-exchanger) with stepwise gradient elution. The isoenzymes originating from small intestine, bone, liver and bile were identified by use of tissue homogenates, pathological sera, and heat inactivation. One isoenzyme was identified in the homogenate of small intestine, two were identified in bone, and two in liver, and biliary and fragment isoenzymes in bile. There are indications that the first liver isoenzyme is derived from the cell membrane and the second liver isoenzyme from the cytosol. The biliary isoenzyme is considered to be a highly sensitive and specific indicator for cholestasis. We propose that the isoenzymes called "Fragmented Isoenzymes" which had been detected in liver homogenate and in bile fluid, represent catabolites of the various isoenzymes in serum.

Transient hyperphosphatasaemia of infancy.

In five patients with benign transient hyperphosphatasaemia (THP), high activities of so-called "atypical" alkaline phosphatase or fragment isoenzymes were detected. One case occurred after rotavirus infection. Incubation with neuraminidase suggested that "atypical" alkaline phosphatase originated from highly glycosylated bone and liver isoenzymes. This may have been due to virus-induced low isoenzyme clearance from serum. The course of isoenzyme activities in THP following rotavirus infection was followed. Determination of atypical alkaline phosphatase may be useful in the diagnosis of THP.

Liquid-chromatographic determination of isoenzymes of alkaline phosphatase in serum and tissue homogenates.

We describe a new method for separating alkaline phosphatase (AP) isoenzymes by means of "high-performance" liquid chromatography. Isoenzymes are eluted from the column (Mono Q HR 5/5, a strong anion-exchanger) with a stepwise gradient of LiCl. The isoenzymes originating from small intestine, bone, liver, and bile were identified by use of tissue homogenates, pathological sera, and heat inactivation.

"Fragmented" isoenzymes of alkaline phosphatase in the diagnosis of transient hyperphosphatasemia.

We describe the appearance of "fragmented" isoenzymes of serum alkaline phosphatase (EC 3.1.3.1) in two cases of transient hyperphosphatasemia. We determined the isoenzymes by liquid chromatography, then characterized them by heat inactivation, inhibition with 5 mmol/L L-phenylalanine solution, and electrophoresis on cellulose acetate membranes. We suspect that a virus-induced decrease in clearance of the enzyme from serum is responsible for a similar increase of bone and liver isoenzyme activities and for the presence of these fragmented isoenzymes in transient hyperphosphatasemia.

Clinical relevance of alkaline phosphatase isoenzyme determinations by high performance liquid chromatography.

A new method for the separation of alkaline phosphatase isoenzymes by means of high performance liquid chromatography (HPLC) is presented. One isoenzyme was identified in homogenate of small intestine, two were identified in bone, and two in liver, and fragment and biliary isoenzymes were identified in bile. Sera from 32 patients with different diseases of the skeletal system or the liver were analysed. High activities of the bone isoenzymes were detected in bone diseases, of the second liver isoenzyme in acute hepatitis and of the first liver and biliary isoenzymes in biliary obstruction. There are indications that the first liver isoenzyme is derived from the cell membrane and the second liver isoenzyme from the cytosol. The biliary isoenzyme is considered to be a highly sensitive and specific indicator for cholestasis.

[Significance and selected examples for using determinations of isoenzyme of alkaline phosphatase in pediatrics]. / Bedeutung und ausgewählte Anwendungsbeispiele von Isoenzymbestimmungen der alkalischen Phosphatase in der Pädiatrie.

The interpretation of total alkaline phosphatase in paediatric patients is complicated by the wide range of normal values in the various age groups. Selected separations of isoenzymes in cases of rickets, hepatitis, cirrhosis, congenital atresia of the bile duct, during cytostatic or anticonvulsant therapy in 32 paediatric patients demonstrate the clinical relevance of isoenzyme determination.

[Establishment of leukaemia cell lines and their significance for clinical and experimental oncology (author's transl)]. / Etablierung von Leukämiezellinien und ihre Bedeutung für die klinische und experimentelle Onkologie.

Permanent in vitro growing leukemic cell lines have been established from all types of immunologically classified childhood leukemias. Essential characteristics of primary blasts and cultured cells are identical. In contrast to lymphoblastoid, non-leukemic cell lines, the Epstein-Barr-virus specific nuclear angiten (EBNA) is not detected. Up to now 8 Non-B-non-T cell lines (6 of them were derived from children with acute lymphoblastic leukemia, 2 from patients with chronic myeloid leukemia), 8 T-lines and one B-line have been established. Three Non-B-non-T lines from children with acute lymphoblastic leukemia (KM-3, RU-3, MH-3) and one T-cell line (JM) were cultivated by ourselves. Cultured blasts represent a pure tumor material which can be propagated in large quantities. Leukemic cell lines reveal a new approach for the search after leukemia-associated proteins and represent another possibility for the experimental investigation of the etiology of leukemia.

[Attempts to enrich and to solubilize the muscarinic acetylcholinereceptor from bovine caudate nucleus (author's transl)]. / Versuche zur Anreicherung und Solubilisierung des muscarinischen Acetylcholinreceptors aus dem Nucleus caudatus vom Rind.

Neuronal membranes of postsynaptic origin twofold enriched in acetylcholinesterase, muscarinic acetylcholinereceptor and (Na+/K+)-ATP-Phosphohydrolase, proteins associated with cholinergic nerve excitability, were prepared with yields between 60 and 75% from bovine caudate nucleus. On subfractionation of these membranes an additional twofold enrichment of the mentioned proteins is achieved in different subfractions. SDS-gradient gel electrophoresis shows that these subfractions have slightly different polypeptide compositions. Neuronal membranes of presynaptic origin on the other hand, prepared from purified synaptosomes, possess only small amounts of the mentioned proteins, showing no enrichment with respect to the homogenate. Solubilization of acetylcholinesterase with 1 M NaC1 as well as of muscarinic acetylcholinreceptor with 2 M NaC1 does not succeed. These proteins are therefore not solely bound by ionic forces to the isolated membranes from bovine caudate nucleus.