Elisabeth Schuller

Rat brain proteins: Two‐dimensional protein database and variations in the expression level

A two—dimensional database of rat brain proteins was constructed. Brain samples from newborn animals were analyzed by two—dimensional electrophoresis and the proteins were identified by matrix‐assisted laser desorption/ionization mass spectrometry. The dababase comprises 210 different proteins, the majority of which are structural components, heat shock proteins and enzymes with various catalytic activities. Several minor differences in the expression level were detected, mainly of quantitative nature, which most likely represent allelic differences. The map may be useful in studies of neurological disorders in animal models of human diseases.

Differences in protein level between neonatal and adult brain

Age is a physiological condition that affects protein expression. We investigated differences in protein level between newborn and adult brains. Brain tissue extracts from male and female adult and neonatal rats were analyzed by two‐dimensional gel electrophoresis. Gel comparison revealed the presence of many differences, of qualitative and quantitative nature, between a neonatal and adult brain. The most significant age‐related difference concerned α‐fetoprotein, which was detected in the brain of neonatal rats only. The levels of 22 proteins, including dihydropyrimidinase‐related proteins 1,3, and 4 and 14‐3‐3 proteins, were higher in the neonatal brain, whereas the levels of 28 proteins, including dihydropyrimidinase‐related protein 2, dynamin‐1 and many enzymes were higher in the adult brain. We did not detect a consistent sex‐related difference in the brain proteins. An inconsistency was observed in the location of the spot representing glial fibrillary acidic protein in the male brain.

Feasibility of Long-Term Intraventricular Therapy with Mafosfamide (n = 26) and Etoposide (n = 11): Experience in 26 Children With Disseminated Malignant Brain Tumors

Treatment options for leptomeningeal disseminated brain tumors are limited by the lack of effective drugs for intrathecal therapy of non-hematologic malignancies. We report on our experience with an intraventricular therapy consisting of mafosfamide, a preactivated cyclophosphamide derivative, and etoposide. Between May 1994 and 2002, 26 patients aged 2–19 years with various intensely pretreated disseminated brain tumors received intraventricular mafosfamide via an indwelling subcutaneous reservoir. Twenty-three of them received a dose of 20mg. Mafosfamide was administered once or twice weekly until remission was achieved and every 2–6 weeks thereafter as maintenance therapy for a total of 736 administrations (2–63/patient). Since March 1998, two patients were switched to receive intraventricular etoposide and nine received etoposide alternating with mafosfamide. Etoposide was given at a dose of 0.5mg×5d every 3–6 weeks for a total of 122 courses (1–29/patient).Immediate toxicities such as transient headaches, nausea, and vomiting occurred with mafosfamide but were manageable with premedication. Etoposide did not cause any discomfort. No long-term toxicities attributable to intrathecal therapy as evidenced by magnetic resonance imaging or neurologic evaluation were observed. Since all patients received some sort of concurrent anti-cancer therapy, the efficacy of intrathecal therapy cannot be assessed independently. However, seven of 13 patients evaluable for response by cerebrospinal fluid (CSF) cytology developed CSF dissemination under systemic chemotherapy and cleared their CSF only after administration of intrathecal mafosfamide. In conclusion, intraventricularly administered mafosfamide at a dose of 20mg and etoposide at a dose of 0.5mg×5d for patients over 2 years of age are feasible and safe and may produce responses.

Brain t-complex polypeptide 1 (TCP-1) related to its natural substrate β1 tubulin is decreased in Alzheimer's disease

The t-complex polypeptide 1 is a selective molecular chaperone in tubulin biogenesis, by that nascent tubulin subunits are bound to t-complex polypeptide 1 and released in assembly competent forms. In neurodegenerative diseases with Alzheimer pathology cytoskeletal proteins are deficient and aggregated. Therefore we examined t-complex polypeptide 1 as represented by the zeta subunit and its specific substrate beta 1 tubulin represented by a truncated product in six brain regions of nine patients with Alzheimers disease, nine patients with Down syndrome and nine controls. We used 2 dimensional electrophoresis with in-gel-digestion and matrix-assisted laser desorption/ ionization- mass spectrometry for the separation and identification of human brain t-complex polypeptide 1 and beta 1 tubulin. When t-complex polypeptide I was related to its natural and specific substrate beta 1 tubulin, the ratio was significantly decreased in the temporal, frontal, parietal cortex and in thalamus of patients with Alzheimers disease. In Down syndrome the t-complex polypeptide 1/beta 1 tubulin ratio was significantly increased in frontal and parietal cortex suggesting a different mechanism for aggregation of microfilament proteins e.g. beta 1 tubulin. Relatively decreased molecular chaperoning of beta 1 tubulin by t-complex polypeptide 1 may lead to misfolded tubulin aggregating and accumulating in plaques and tangles, a hallmark of Alzheimers disease. Our contribution provides first clues for a mechanism of microtubular accumulation in Alzheimers disease and challenges further studies on different chaperones and chaperonins in the brain of patients with neurodegenerative diseases.

Intrathecal mafosfamide therapy for pediatric brain tumors with meningeal dissemination

The treatment of childhood brain tumors with cerebrospinal fluid (CSF) dissemination is limited by the relative inaccessibility of the CSF to drugs administered systemically and the paucity of available agents for intrathecal therapy. Mafosfamide is a cyclophosphamide derivative, which does not require hepatic activation and thus can be utilized for regional therapy. Between May 1994 and December 1996, 16 patients 2 to 19 (median 12) years old with various disseminated brain tumors were treated with intraventricular mafosfamide via an indwelling subcutaneous reservoir. The patients received mafosfamide at a dose of 20 mg once or twice weekly until remission was achieved, followed by weekly administrations as consolidation therapy, and every 3 to 4 weeks thereafter for maintenance therapy. Except for transient headaches, nausea and vomiting during and immediately after mafosfamide administration no toxicities were observed. Nine of the 16 patients were evaluable for response by CSF cytology. Eight had complete responses and one patient did not respond. In addition to mafosfamide all patients received systemic chemotherapy as well. However, 4 of the 8 responding patients had developed CSF dissemination under concurrent systemic therapy and cleared their CSF only after administration of intrathecal mafosfamide. At a median follow-up of 21 months, 7 patients are in complete and 4 in partial remission, 2 have stable disease and 3 died of tumor progression. We conclude that mafosfamide at a dose of 20 mg can be safely administered into the CSF and may produce responses and prolong remission of the leptomeningeal disease.

Evidence against increased glycoxidation in patients with Alzheimer's disease

Neuropathological findings of Alzheimers disease (AD) are intracellular (neurofibrillary tangles) and extracellular (senile plaques) filamentous protein aggregates. Non-enzymatic glycation has been proposed as a primary factor in this pathogenesis, leading to increased insolubility of tau protein and beta-amyloid. The aim of our study was to test the hypothesis that increased glycoxidation, i.e. increased levels of oxidized products from non-enzymatic glycation could be found in brains of patients with AD and of aged Down syndrome (DS) subjects with abundant AD-like neuropathological lesions. Frontal cortex specimens were assayed for pentosidine (Pent) and N-epsilon-carboxymethyl-lysine (CML) by reversed phase high performance liquid chromatographical methods. Pent and CML levels in AD (n = 10; Pent, 35.5 +/- 4.84 mumol/g wet-weight tissue; CML, 135.2 +/- 5.0 mumol/g wet-weight tissue) were comparable to DS (n = 9; Pent, 36.4 +/- 3.21; CML, 133.5 +/- 4.7) and controls (n = 10; Pent, 35.2 +/- 3.55; CML, 136.9 +/- 3.3). We conclude that the results are not compatible with the concept of increased glycoxidation in AD compared to normal aging.

Genes involved in the pathophysiology of perinatal asphyxia

Mechanisms in the pathogenesis of perinatal asphyxia (PA) at the gene level are only beginning to be elucidated, although gene hunting using differential display has revealed differences in gene expression between hypoxic and normoxic cells in vitro. As no information on gene expression was available from in vivo studies, we decided to use a non-invasive and clinically relevant animal model of PA for mRNA hunting applying the subtractive hybridization method. mRNAs from normoxic rat brain and brain of rat pups with 20 min of asphyxia were isolated and compared by this technique. The resulting subtracted mRNAs were converted to cDNA, sequenced and identified by gene bank data. A series of transcripts representing transcription factors, transporters, metabolic factors, were found to be up- or downregulated providing insight into mechanisms of PA, and on the other hand, genes with unknown functions could be given a preliminary role i.e. in PA. Results obtained with this powerful tool are now challenging quantitative determination of these genes and gene products at the protein and activity level to confirm their role in PA.

Downregulation of the transcription factor scleraxis in brain of patients with Down Syndrome

Performing gene hunting in fetal Down Syndrome (DS) brain, we found a downregulated sequence with 100% homology to the basic-helix-loop-helix transcription factor (TF) scleraxis (Scl). It was the aim of the study to evaluate Scl-mRNA steady state levels in adult DS brain with Alzheimers disease (AD) neuropathological changes, brain of patients with AD, and controls in order to find out whether Scl-downregulation is linked to DS per se or simply to neurodegeneration, common to both disorders. Determination of Scl-mRNA steady state levels was carried out by a blotting method in frontal, parietal, temporal, occipital lobe and cerebellum. We found significantly decreased Scl-transcripts in brain of DS and AD, both, when normalized versus the house-keeping gene beta actin or total RNA. We demonstrate the significant decrease of Scl-mRNA steady state levels in the pathogenesis of DS and AD suggesting a tentative role for this transcription factor in the development of the neurodegenerative processes known to occur in both disorders. More specifically, the biological meaning of the downregulation of Scl may be the involvement in the pathogenesis of impaired neuronal plasticity and wiring observed in DS and AD, phenomena regulated by the concerted action of the many transcription factors expressed in human brain.

Apolipoprotein (a) Levels and Atherogenic Lipid Fractions in Relation to the Degree of Urinary Albumin Excretion in Type 2 Diabetes mellitus

The relationship between serum lipids, apolipoprotein levels including apolipoprotein (a) to albumin excretion rate (AER) in 52 (17 male, 35 female) type 2 diabetic patients was studied. Patients were classified in groups as follows: (1) patients with normal AER (< 30 mg/24 h); (2) patients with microalbuminuria (AER 30-300 mg/24 h), and (3) patients with macroalbuminuria (AER > 300 mg/24 h). Apolipoprotein B levels were significantly (p < 0.05) elevated in the patients with macroalbuminuria compared to the normoalbuminuric group. Further, we observed a significant correlation between total cholesterol, low-density lipoprotein cholesterol, as well as apolipoprotein B levels to the degree of AER. No correlation between apolipoprotein (a) levels and AER, which is in contrast to recently published data for type 1 diabetic patients, was found. Systolic blood pressure was significantly higher (p < 0.002) in the patients with micro- and macroalbuminuria compared to the group with normal albumin excretion, whereas no relationship between diastolic blood pressure and albuminuria was found. We conclude that elevated increased atherogenic lipid fractions in addition to systolic hypertension may contribute to an increased cardiovascular risk in type 2 diabetic patients with proteinuria.

Prolonged second response to cisplatin, etoposide, and ifosfamide in a child with a recurrent brainstem glioblastoma

The prognosis for patients with malignant brain stem tumors is poor. The authors report on a 6-year-old girl with a biopsy proven pontine glioblastoma, who, after initial chemo-radiotherapy and tumor progression, experienced a prolonged second response to a salvage therapy consisting of cisplatin, etoposide, and ifosfamide. The patient recovered from her life-threatening condition with almost complete resolution of all neurologic deficits paralleled by a dramatic shrinkage of the tumor documented by magnetic resonance imaging.