Temporal expression of inflammatory mediators in brain basilar artery vasculitis and cerebrospinal fluid of rabbits with coccidioidal meningitis.

Strokes due to transmural vasculitis associated with coccidioidal meningitis result in significant morbidity and mortality. The immunological and inflammatory processes responsible are poorly understood. To determine the inflammatory mediators, i.e. cytokines, chemokines, iNOS, matrix metalloproteinase-9 (MMP-9), that possibly contribute to vasculitis, temporal mRNA expression in brain basilar artery samples and MMP-9 protein in the CSF of male NZW rabbits infected intracisternally with 6.5 x 10(4) arthroconidia of Coccidioides immitis were assessed. Five infected and 3 sham-injected rabbits at each time point were euthanized 4, 9, 14 and 20 days post infection. All infected rabbits had neurological abnormalities and severe vasculitis in the basilar arteries on days 9-20. In basilar arteries of infected animals versus controls, mRNAs encoding for IL-6, iNOS, IFN-gamma, IL-2, MCP-1, IL-1beta, IL-10, TNF-alpha, CCR-1, MMP-9, TGF-beta, as well as MMP-9 protein in CSF, were found to be significantly up-regulated. Thus, this study identified inflammatory mediators associated with CNS vasculitis and meningitis due to C. immitis infection. Assessment of the individual contribution of each mediator to vasculitis may offer novel approaches to the treatment of coccidioidal CNS infection. This study also provides unique methodology for immunology studies in a rabbit model.

Modulation by canine interferon-gamma of major histocompatibility complex and tumor-associated antigen expression in canine mammary tumor and melanoma cell lines.

In an effort to enhance the antigenicity of canine tumor cells, canine interferon-gamma (CnIFN-gamma) was applied in vitro to seven canine mammary tumor (CMT) and two canine melanoma (CML) cell lines. Surface expression of major histocompatibility complex (MHC) antigens and tumor-associated antigens (TAA) was measured by a flow cytometric fluorescence assay using commercially available anti-MHC antibodies, and anti-canine TAA monoclonal antibodies generated against CMT and CML cell lines. Compared to constitutive antigen levels in untreated cells, treatment with CnIFN-gamma resulted in increased expression of MHC class I and II antigens (up to 19- and 167-fold, respectively) and a TAA (up to 5-fold) by CMT cell lines, and increased expression of class I antigen (131-fold) by one CML and of class II antigen (18-fold) by the other CML cell line. Expression of MHC antigens and a TAA by tumor cells was increased by Cn-IFN-gamma treatment, and such an increase may be of potential benefit in tumor cell recognition and rejection by the immune system.

Correlation of RFLP typing and MLC reactivity in dogs.

The human recombinant HLA-DRB1 gene probe was used for histocompatibility typing of two families of beagles for the DLA-D equivalent by using restriction fragment length polymorphisms (RFLP). This method was able to determine the segregation of these genes from the parental animals to the individual F1 offspring. Mixed lymphocyte culture (MLC) reactivity as well as serological typing for class I histocompatibility antigens were also performed for comparison. It was found that there was a high correlation between these three methods. We therefore conclude that RFLP typing is an effective procedure for predicting MLC reactivity in dogs and propose that it is a suitable genotyping method for assignment of class II antigen compatibility for donor-recipient pairs in conjunction with organ transplant studies.

Restoration of binding of oxidized transcription factor IIIA to 5S RNA by thioredoxin.

7S particles from Xenopus oocytes were completely dissociated under non-reducing conditions. Studies using glycerol gradient centrifugation show that unlike the native 7S particle in which 5S RNA and TFIIIA co-sedimented in a fairly sharp peak, the RNA from the denatured 7S sedimented at the position corresponding to the 5S RNA and the TFIIIA sedimented as a wide peak between 6S and 12S. Thioredoxin from E. coli can catalyze the reactivation of the TFIIIA as measured by its ability to reform the 7S particle. The rate of reactivation with thioredoxin was significantly greater than with dithiothreitol. Oxidized thioredoxin was unable to reactivate TFIIIA. Pure TFIIIA can be inactivated and subsequently reactivated in the same way by formation of a cross-linked structure via intermolecular disulfide bridges.

Selective phosphorylation of human DNA methyltransferase by protein kinase C.

Human DNA methyltransferase, the enzyme thought to be responsible for the somatic inheritance of patterns of DNA methylation, is an effective substrate for phosphorylation by protein kinase C. This provides a plausible mechanistic link between the action of tumor promoting phorbol esters, which stimulate protein kinase C, and abnormal patterns of DNA methylation often observed in transformed cells.

Purification of human DNA (cytosine-5-)-methyltransferase.

We have developed a facile procedure for the purification of DNA methyltransferase activity from human placenta. The procedure avoids the isolation of nuclei and the dialysis and chromatography of large volumes. A purification of 38,000-fold from the whole cell extract has been achieved. The procedure employs ion exchange, affinity, and hydrophobic interaction chromatography coupled with preparative glycerol gradient centrifugation. A protein of 126,000 daltons was found to copurify with the activity and was the major band seen in the most highly purified material after SDS gel electrophoresis. This observation, coupled with an observed sedimentation coefficient of 6.3S, suggests that the enzyme is composed of a single polypeptide chain of this molecular weight. Hemimethylated DNA was found to be the preferred substrate for the enzyme at each stage in the purification. The ratio of the activity of the purified product on hemimethylated to that on unmethylated M13 duplex DNA was about 12 to 1. Thus, the purified activity has the properties postulated for a maintenance methyltransferase. The availability of highly purified human DNA methyltransferase should facilitate many studies on the structure, function, and expression of these activities in both normal and transformed cells.