Point mutation causing constitutive signaling of CXCR2 leads to transforming activity similar to Kaposi's sarcoma herpesvirus-G protein-coupled receptor.

The chemokine receptor CXCR2 is the closest homologue to Kaposi's sarcoma herpesvirus-G protein-coupled receptor (KSHV-GPCR), which is known to be constitutively activated and able to cause oncogenic transformation. Among G protein-coupled receptors, a DRY sequence in the second intracellular loop is highly conserved. However, the KSHV-GPCR shows a VRY sequence instead. In this study, we exchanged Asp138 of the DRY sequence in the CXCR2 with a Val (D138V), the corresponding amino acid in KSHV-GPCR, or with a Gln (D138Q), and investigated the functional consequences of these mutations. In focus formation and soft agar growth assays in NIH 3T3 cells, the D138V mutant exhibited transforming potential similar to the KSHV-GPCR. Surprisingly, the CXCR2 wild type itself showed transforming activity, although not as potently, due to continuous autocrine stimulation, whereas the D138Q mutant formed no foci. In agreement with these results were high levels of inositol phosphate accumulation in the D138V mutant and the KSHV-GPCR, indicating constitutive activity. These data emphasize the importance of the DRY sequence for G protein-coupled signaling of the CXCR2. Either constitutive activation or persistent autocrine stimulation of the CXCR2 causes transformation similar to KSHV-GPCR-transfected cells, probably activating the same signal transduction cascade that can abrogate normal growth control mechanisms.     

Cell-mediated immunity in experimental allergic encephalomyelitis: cross reactivity between myelin basic protein and mycobacteria antigens.

Guinea pigs injected with Freund's incomplete adjuvant emulsified with guinea pig spinal cord, purified guinea pig myelin basic protein, or human myelin basic protein showed dermal reactivity to both of the basic proteins as well as to mycobacteria antigens. Animals receiving only mycobacteria antigens expressed dermal reactivity to the sensitizing antigen in addition to basic protein. This cross reactivity may help explain the role of mycobacteria in inducing and protecting against EAE, and may have important implications concerning human demyelinating diseases.     

All KLH preparations are not created equal

Keyhole limpet hemocyanin (KLH) preparations used for dermal testing in human volunteers may contain factors which elicit false positive reactions. These factors can be associated with the hemolymph collection procedure and possibly to a change in collection beds by the limpet supplier. Rapid collection of hemolymph from animals obtained from specified limpet beds resulted in KLH which produced negligible dermal reactivity in normal individuals.     

The species-specific cell-binding site of the aggregation factor from the sponge Microciona prolifera is a highly repetitive novel glycan containing glucuronic acid, fucose, and mannose

Species-specific adhesion of dissociated cells from the marine sponge Microciona prolifera is mediated by a Mr = 2 x 10(7) proteoglycan-like aggregation factor (MAF) via two highly polyvalent functional domains, a cell-binding and a self-interaction domain. Glycopeptide N-glycosidase F release of a major glycan of Mr = 6.3 gamma 10(3) (G-6) from the MAF protein core resulted in the loss of cell binding activity, indicating a role of this polysaccharide molecule in MAF-cell association. The G-6 glycan was isolated and purified after complete Pronase digestion of MAF using gel electrophoresis, gel filtration, and ion exchange chromatography. Quantification of the amount of carbohydrate recovered in G-6 showed that one MAF molecule has about 950 repeats of this glycan. In its monomeric state G-6 did not display any measurable binding to cells (K alpha less than or equal to 10(3) M-1). Intermolecular cross-linking of the G-6 glycan with glutaraldehyde resulted, however, in the concomitant recovery of polyvalency (about 2200 repeats of G-6 per polymer of Mr greater than or equal to 1.5 x 10(7) and species-specific high cell binding affinity (K alpha = 1.6 x 10(9) M-1) but not of the MAF-MAF self-interaction activity. Thus, the G-6 glycan is the multiple low affinity cell-binding site involved in cell-cell recognition and adhesion of sponge cells. The G-6 glycan has 7 glucuronic acids, 3 fucoses, 2 mannoses, 5 galactoses, 14 N-acetylglucosamines, 2 sulfates, and 1 asparagine. Such a unique chemical composition indicates a new type of structure which includes features of glycosaminolycans and N-linked polysaccharides.     

Evaluation of UVB reduction by materials commonly used in reptile husbandry

Ultraviolet B (UVB) irradiation (285–320 nm) is considered important for metabolic processes and reproduction in many reptile species by facilitating the synthesis of vitamin D₃. In captivity, UVB radiation reaching an animal may be diminished by the properties of the materials used in enclosure construction. We investigated the UVB‐attenuating properties of 14 materials commonly used in cage tops for reptile enclosures. Irradiances were measured by two types of hand‐held broadband radiometers and the D₃‐synthesizing potential was assessed by the use of an in vitro model. For UV‐transmitting acrylic, a significant discrepancy between meter irradiances and in vitro model values for D₃‐synthesizing ability was observed, with meter readings underestimating the blocking effect. In contrast, attenuation of UVB irradiances by air‐permeable materials, such as wire screen, measured with meters was generally comparable to the attenuation of D₃‐synthesizing ability as measured by in vitro models. Relatively simple meter readings can therefore be used to reflect reduction of D₃‐synthesizing ability through air‐permeable materials. Zoo Biol 26:417–423, 2007. © 2007 Wiley‐Liss, Inc.     

Involvement of carbohydrates as multiple low affinity interaction sites in the self-association of the aggregation factor from the marine sponge Microciona prolifera

Cell aggregation in the marine sponge Microciona prolifera is mediated by a multimillion molecular weight aggregation factor (MAF) and is based on two functional properties, a Ca2+-independent cell binding activity and a Ca2+-dependent factor-factor self-interaction. Monoclonal antibodies were prepared against purified MAF, and one clone was characterized which selectively inhibited the MAF-MAF association activity. Binding of the blocking antibody (Block 1) to MAF demonstrated that this epitope structure was present in 1100 copies per one MAF molecule of Mr = 2 X 10(7). Such blocking antibodies precipitated a small molecular weight protein-free glycan fraction prepared from MAF by Pronase digestion, thus indicating that the highly repetitive epitope is located in the carbohydrate portion of the molecule. Since the inhibitory activity of the Block 1 antibody could only be achieved when most of the sites were occupied by Fab fragments of this antibody, the self-association of MAF seemed to be based on the polyvalency of the carbohydrate determinants. The affinity of the protein-free glycans was very low as shown by the fact that they did not display any measurable self or MAT binding activity in their monomeric form. After cross-linking them with glutaraldehyde into polymers of the size of MAF, however, the self-interaction could be reconstituted. Thus, the MAF-MAF association activity, which is needed for cell aggregation in sponges, seems to be based on multiple low affinity carbohydrate-carbohydrate interactions, which is different from most interactions mediated by adhesion molecules characterized so far.     

Nicotinamide: suppression of lymphocyte transformation with a component identified in human transfer factor.

The component in human transfer factor (TF) (Fraction IV, from exclusion chromatography on Sephadex G-25) responsible for suppression of antigen-induced lymphocyte transformation was previously identified as nicotinamide. Commercially available nicotinamide was subsequently shown to produce suppression of antigen-induced responses in vitro previously observed with TF Fraction IV. Nicotinamide was found to be nontoxic at the highest concentrations employed (10(-2)M) and suppressive over a relatively broad range (10(-5) to 10(-2)M. The suppression appeared to be related to the magnitude of antigen- or mitogen-induced transformation and was apparent even when nicotinamide was added as late as 48 hr after stimulant addition.