Viral interleukin-6 encoded by rhesus macaque rhadinovirus is associated with lymphoproliferative disorder (LPD).

BACKGROUND: Rhesus macaques (RM) co-infected with simian immunodeficiency virus (SIV) and rhesus macaque rhadinovirus (RRV) develop abnormal cellular proliferations characterized as extra-nodal lymphoma and retroperitoneal fibromatosis (RF). RRV encodes a viral interleukin-6 (vIL-6), much like Kaposi's sarcoma-associated herpesvirus, and involvement of the viral cytokine was examined in proliferative lesions. METHODS: Formalin fixed tissue from RM co-infected with SIV and RRV were analyzed for RRV genomes by in situ hybridization and RRV vIL-6 expression by immunofluorescence analysis. RESULTS: In situ hybridization analysis indicated that RRV is present in both types of lesions. Immunofluorescence analysis of different lymphomas and RF revealed positive staining for vIL-6. Similarly to KS, RF lesion is positive for vimentin, CD117 (c-kit), and smooth muscle actin (SMA) and contains T cell, B cell and monocytes/macrophage infiltrates. CONCLUSIONS: Our data support the idea that vIL-6 may be critical to the development and progression of lymphoproliferative disorder in RRV/SIV-infected RM.

Uncertainty in rate loss corrections based on counting a periodic pulser.

Tomographic gamma scanning of waste produces three-dimensional transmission and emission images. These are used to derive item-specific attenuation correction factors that improve the accuracy of non-destructive waste assay. For each vertical layer, data grabs of short duration are acquired as the waste item is rotated and translated. The image reconstruction demands accurate rate loss corrections to minimize assay bias. For this application a pulser was used to perform the necessary rate loss corrections. In this work, we summarize the benefits of the pulser approach and review the basic principles on which the method is based. We extend the treatment to include a derivation of the expression for the uncertainty in the net pulser peak area in the presence of an underlying continuum. We report experimental results, taken using a Canberra model WM2900 Tomographic Gamma Scanner, over a broad range of count-rates and peak-to-continuum ratios. Repeat counts under controlled conditions allowed the correction factor and its variance to be determined and compared against expectations. These results confirm the validity of the correction factor formula and the corresponding expression for its uncertainty. The rate loss analysis has been built into a Monte Carlo Replicate engine to allow the uncertainty to be propagated into the total measurement uncertainty of the final assay.

Palmitylation of neuromodulin (GAP-43) is not required for phosphorylation by protein kinase C.

Neuromodulin (also designated GAP-43, B-50, and F-1) is a prominent protein kinase C substrate attached to the membranes of neuronal growth cones during development and to presynaptic membranes in discrete subsets of adult synapses. In this study, we have examined the relationship between the attachment of neuromodulin to membranes and its phosphorylation by protein kinase C. To address this issue, we have compared wild-type and mutant neuromodulins expressed in cells that normally lack the protein. Wild-type neuromodulin expressed in Chinese hamster ovary cells was associated with membranes, incorporated [3H]palmitic acid, and was phosphorylated in response to phorbol ester treatment. Substitution of serine 41, the in vitro protein kinase C site, abolished the phorbol ester response, indicating that serine 41 serves as the sole protein kinase C phosphorylation site in vivo. Substitution of the putative fatty acylation sites, cysteines 3 and 4, abolished membrane association as well as [3H]palmitic acid labeling of neuromodulin. Fatty acylation therefore appears to serve as the mechanism for anchoring neuromodulin to membranes. Surprisingly, the soluble cysteine substitution mutant was phosphorylated by protein kinase C at a rate indistinguishable from that of the wild-type protein. Therefore, membrane association may not be required for the phosphorylation of neuromodulin by protein kinase C.

Expression of cDNAs encoding wild-type and mutant neuromodulins in Escherichia coli: comparison with the native protein from bovine brain.

Murine cDNA that encodes neuromodulin, a neurospecific calmodulin binding protein, was inserted into the plasmid pKK223-3 for expression in Escherichia coli. After being transformed into E. coli strain SG20252 (lon-), the expression vector directed the synthesis of a protein that was recognized by polyclonal antibodies raised against bovine neuromodulin. The recombinant protein expressed in E. coli was found to be tightly associated with insoluble cell material and was extractable only with guanidine hydrochloride or sodium dodecyl sulfate. Following solubilization with guanidine hydrochloride, the protein was purified to apparent homogeneity by a single CaM-Sepharose affinity column step with a yield of 0.2 mg of protein/L of E. coli culture. The availability of the purified recombinant neuromodulin made it possible to answer several specific questions concerning the structure and function of the protein. Despite the fact that murine neuromodulin is 12 amino acid residues shorter than the bovine protein and the recombinant protein expressed in E. coli may lack any posttranslational modifications, the two proteins displayed similar biochemical properties in almost all respects examined. They both had higher affinity for CaM-Sepharose in the absence of Ca2+ than in its presence; they were both phosphorylated in vitro by protein kinase C in a Ca2+- and phospholipid-dependent manner; neither form of the proteins was autophosphorylated, and the phosphorylated form of the proteins did not bind calmodulin. The recombinant neuromodulin and neuromodulin purified from bovine brain had similar, but not identical, affinities of calmodulin, indicating that the palmitylation of the protein that occurs in animal cells is not crucial for calmodulin interactions.(ABSTRACT TRUNCATED AT 250 WORDS)