Persistence of psychosine in brain lipid rafts is a limiting factor in the therapeutic recovery of a mouse model for Krabbe disease.

Sphingolipids are intrinsic components of membrane lipid rafts. The abnormal accumulation of these molecules may introduce architectural and functional changes in these domains, leading to cellular dysfunction. Galactosylsphingosine (psychosine) is a pathogenic lipid raft-associated molecule whose accumulation leads to brain deterioration and irreversible neurological handicap in the incurable leukodystrophy Krabbe disease (KD). The relevance of clearing excessive levels of pathogenic psychosine from lipid rafts in therapy for KD has not been investigated. The work presented here demonstrates that psychosine inhibits raft-mediated endocytosis in neural cells. In addition, although in vitro enzyme reconstitution is sufficient for the reversal of related endocytic defects in affected neural cells, traditional in vivo enzyme therapies in the mouse model of KD appear to be insufficient for complete removal of pathogenic levels of raft-associated psychosine. This work describes a mechanism that may contribute to limiting the in vivo efficacy of traditional therapies for KD.

Pulmonary adiaspiromycosis in critically endangered northern hairy-nosed wombats (Lasiorhinus krefftii).

BACKGROUND: The northern hairy-nosed wombat (Lasiorhinus krefftii) is critically endangered, with only 200 individuals remaining in the wild. Individuals are rarely available for detailed pathological assessment and identification of disease threats to individuals is critically important to species conservation. CASE REPORT: Two male northern hairy-nosed wombats, part of the Richard Underwood Nature Refuge population, were presented for necropsy, 5 months apart. They were found to have succumbed to adiaspiromycosis caused by the fungus Emmonsia parva. Pathological presentations were of severe pulmonary oedema and fibrosis, and pleuritis, respectively. Characteristic fungal adiaspores were noted on histopathological examination. The wombats had concurrent variably severe ectoparasite and endoparasite burdens. CONCLUSION: These are the first reported cases of adiaspiromycosis in northern hairy-nosed wombats and the organism was associated with significant pathological changes. The rarity and the logistical challenges of presenting northern hairy-nosed wombats for pathological assessment are a challenge to identifying disease threats in this critically endangered species.

Intracellular Localization and intercellular heterogeneity of the human DNA repair protein O(6)-methylguanine-DNA methyltransferase.

O(6)-Methylguanine-DNA methyltransferase (MGMT) is a DNA repair protein that removes alkyl adducts from DNA and may be important in tumor resistance to alkylation chemotherapy. MGMT was visualized in human cells and tumor tissues with monoclonal antibodies against MGMT and immunofluorescence microscopy, and fluorescent signals were quantified by digital image analysis. MGMT was found both in the cytoplasm and the nucleus, and in either locale the protein reacts with alkylated DNA bases and becomes inactivated and lost from the cell. Cell lines in culture and xenografts showed a broad normal distribution of nuclear MGMT levels, but human brain tumors often showed a skewed distribution, with a significant fraction of cells with high levels of MGMT. O(6)-Benzylguanine, a suicide substrate inactivator for MGMT activity, reduced MGMT in human cells and in a mouse xenograft to levels undetectable by antibody assay 1 h post-treatment. In melanoma specimens taken from a patient 3 h post-treatment with temozolomide, MGMT levels were reduced by 70%. This quantitative immunofluorescence assay can be used to monitor MGMT and it depletion in human tumors to improve the use of alkylating agents in cancer chemotherapy.

Analysis of O6-methylguanine-DNA methyltransferase in individual human cells by quantitative immunofluorescence microscopy.

A quantitative assay of immunofluorescence is described that can be performed on individual cells from standard pathologic specimens using fluorescence microscopy. The technique has been applied to measurement of O6-methylguanine-DNA methyltransferase, a DNA repair protein that is a molecular marker for resistance to chloroethylnitrosources used in cancer chemotherapy. The immunofluorescence assay makes use of monoclonal antibodies with specificity for human transferase, fluorescence microscopy with digital imaging, fluorescent bead internal standards, and computerized image analysis. This method is specific for the transferase, produces results correlated with activity measurements, and yields new data about tissue heterogeneity and subcellular localization previously unavailable with standard assay methods.

The signature 10-hydroxy stearic acid thought to correlate with infectivity in oocysts of Cryptosporidium species is an artifact.

Heating or freezing leads to loss in infectivity of oocysts of Cryptosporidium parvum toward neonatal BALB/c mice and is reflected in the profile of the polar lipid fatty acids. Upon loss of infectivity, the ratio of polar lipid to neutral lipid fatty acid decreased and the relative proportions of 18:1n-9 also decreased; proportions of 18:2n-6 and 20:5n-6 increased, whereas the proportions of 16:0 remained constant with freezing. During these investigations, a novel fatty acid, 10-OH 18:0, was discovered in the glycolipid fraction. The identification of a fatty acid unique to species of Cryptosporidium was thought to provide a specific biomarker for this organism. Cryptosporidium also demonstrated fluctuations in absolute quantities of 10-OH 18:0 with events that lead to loss of infectivity. This led to the presumed correlation of this biomarker with infectious Cryptosporidium. The 10-OH 18:0 was putatively localized at the sn-2 position of phosphatidylethanolamine. High-performance liquid chromatography/electrospray ionization mass spectrometry revealed that the 10-OH 18:0 existed principally in the free fatty acid form. Herein, we establish that the free fatty acid 10-OH 18:0 was, in actuality, an artifact of the procedures for sample preparation.

Structural polypeptides of California encephalitis virus: BFS-283.

The polypepetides of California encephalitis virus (BFS-283) were analyzed by polyacrylamide gel electrophoresis (PAGE). Four polypeptides were detected in virions grown in both BHK-21 and LLC-MK2 cell cultures with molecular weights of 17,500. 30,000, 38,000, and 82,000 (VP-1, VP-2, VP-3, and VP-4, respectively). Viral proteins 2, 3, and 4 were glycoproteins and appeared to be associated with the envelope of the virus. Treatment of virions (rho=1.18 g/cm3) with then non-ionic detergent, NP-40, allowed detection of a RNA-rich fraction (rho=1.26/cm3) with contained the smallest polypeptides (VP-1).