Two-Step Enzymatic Modification of Solid-Supported Bergenin in Aqueous and Organic Media.

The natural flavonoid bergenin was directly immobilized onto carboxylic acid functionalized controlled pore glass (carboxy-CPG) at 95% yield. Immobilized bergenin was brominated via chloroperoxidase in aqueous solution and then transesterified with vinyl butyrate in diisopropyl ether by subtilisin carslberg (SC) extracted into the organic solvent via ion pairing. Enzymatic cleavage of 7-bromo-4-butyrylbergenin from carboxy-CPG (9.6% final yield) was accomplished using lipase B (LipB) in an aqueous/organic mixture (90/10 v/v of water/acetonitrile), demonstrating the feasibility of solid phase biocatalysis of a natural product in aqueous and non-aqueous media.

Delivery of temozolomide to the tumor bed via biodegradable gel matrices in a novel model of intracranial glioma with resection.

INTRODUCTION: We have completed in vivo safety and efficacy studies of the use of a novel drug delivery system, a gel matrix-temozolomide formulation that is injected intracranially into the post-resection cavity, as a candidate for glioma therapy. METHODS: A rat intracranial resection model of C6-GFP intracranial glioma was used for safety and toxicity studies. Biodistribution studies were performed using gel matrix-gallocyanine formulations and were evaluated at various time intervals using real-time analysis of dye distribution. Additionally, the resection model was used to determine the efficacy of gel matrix-temozolomide as compared to blank gel matrix. A subcutaneous human xenograft glioma model was used to further assess the efficacy of gel matrix-temozolomide in reducing the overall tumor load. RESULTS: Gel matrix-temozolomide exhibited minimal cytotoxicity toward normal brain tissue while displaying high levels of oncolytic activity toward glioma cells. In the intracranial glioma resection and subcutaneous glioma model, administration of gel matrix-temozolomide directly to the tumor bed was well tolerated and effective at reducing the tumor load. A significant reduction of tumor load was observed (P < 0.0001) in the 30% temozolomide group (approximately 95%) as compared to blank control. There was little morbidity and no mortality associated with gel matrix treatment. CONCLUSIONS: Gel matrix-temozolomide appears to be safe and effective when used in vivo to treat intracranial glioma and warrants further development as a potential adjuvant therapy.

cDNA cloning and characterization of a novel gene encoding the MLF1-interacting protein MLF1IP.

Myelodysplasia/acute myeloid leukemia (MDS/AML) is characterized by a t(3;5)(q25.1;q34) chromosomal translocation that forms a fusion gene between nucleophosmin (NPM) and MDS/myeloid leukemia factor 1 (MLF1). We identified a novel protein, MLF1-interacting protein (MLF1IP), that specifically associates with MLF1 by yeast two-hybrid analysis and in pulldown assays, and colocalizes with it in both the nuclei and cytoplasm of cells. The MLF1IP gene locus is at chromosome 4q35.1 and is composed of 14 exons spanning 75.8 kb of genomic DNA. The MLF1IP cDNA encodes a 46-kDa protein that contains two bipartite and two classical nuclear localization signals, two nuclear receptor-binding motifs (LXXLL), two leucine zippers, two PEST residues and several potential phosphorylation sites. MLF1IP transcripts are expressed in a variety of tissues (e.g. fetal liver, bone marrow, thymus and testis). MLF1IP appears to be a lineage-specific gene whose expression is confined exclusively to the CFU-E erythroid precursor cells, but not in mature erythrocytes. These observations, together with previous data demonstrating a role for MLF1 in suppressing red cell maturation, suggest a possible role for MLF1IP and MLF1 deregulation in the genesis of erythroleukemias.

Regulation of myeloid leukemia factor-1 interacting protein (MLF1IP) expression in glioblastoma.

The myelodysplasia/myeloid leukemia factor 1-interacting protein MLF1IP is a novel gene which encodes for a putative transcriptional repressor. It is localized to human chromosome 4q35.1 and is expressed in both the nuclei and cytoplasm of cells. Northern and Western blot analyses have revealed MLF1IP to be present at very low amounts in normal brain tissues, whereas a number of human and rat glioblastoma (GBM) cell lines demonstrated a high level expression of the MLF1IP protein. Immunohistochemical analysis of rat F98 and C6 GBM tumor models showed that MLF1IP was highly expressed in the tumor core where it was co-localized with MLF1 and nestin. Moreover, MLF1IP expression was elevated in the contralateral brain where no tumor cells were detected. These observations, together with previous data demonstrating a role for MLF1IP in erythroleukemias, suggest a possible function for this protein in glioma pathogenesis and potentially in other types of malignancies.

Ataxia.

Ataxia is a disorder of balance and coordination resulted from dysfunctions involving cerebellum and its afferent and efferent connections. While a variety of disorders can cause secondary ataxias, the list of genetic causes of ataxias is growing longer. Genetic abnormalities may involve mitochondrial dysfunction, oxidative stress, abnormal mechanisms of DNA repair, possible protein misfolding, and abnormalities in cytoskeletal proteins. Few ataxias are fully treatable while hope for efficacious gene therapy and pharmacotherapy is emerging. A discussion of the ataxias is presented here with brief mention of acquired ataxias, and a greater focus on inherited ataxias.

Subclinical rhythmic electrographic discharges of adults in a patient with migraine.

Subclinical rhythmic electrographic discharges of adults (SREDA) is a distinctive electroencephalogram (EEG) pattern seen in adults and is thought to have little clinical significance. These patterns are generally considered nonspecific. We evaluated a 45-year-old female patient with migraine in whom SREDA seemed to have a temporal relation to the migraine symptoms of the patient.

Direct solubilization of enzyme aggregates with enhanced activity in nonaqueous media.

A protein solubilization method has been developed to directly solubilize protein clusters into organic solvents containing small quantities of surfactant and trace amounts of water. Termed "direct solubilization," this technique was shown to solubilize three distinct proteins - subtilisin Carlsberg, lipase B from Candida antarctica, and soybean peroxidase - with much greater efficiencies than extraction of the protein from aqueous solution into surfactant-containing organic solvents (referred to as extraction). More significant, however, was the dramatic increase in directly solubilized enzyme activity relative to extracted enzyme activity, particularly for subtilisin and lipase in polar organic solvents. For example, in THF the initial rate towards bergenin transesterification was ca. 70 times higher for directly solubilized subtilisin than for the extracted enzyme. Furthermore, unlike their extracted counterparts, the directly solubilized enzymes yielded high product conversions across a spectrum of non-polar and polar solvents. Structural characterization of the solubilized enzymes via light scattering and atomic force microscopy revealed soluble proteins consisting of active enzyme aggregates containing approximately 60 and 100 protein molecules, respectively, for subtilisin and lipase. Formation of such clusters appears to provide a microenvironment conducive to catalysis and, in polar organic solvents at least, may protect the enzyme from solvent-induced inactivation.