Divergent patterns of selection on the DAB and DXB MHC class II loci in Xiphophorus fishes.

Two MHC class II loci, DAB (a classical class II locus) and DXB (putatively a non-classical class II locus), were sequenced in samples of individuals from two populations of swordtail fish, Xiphophorus multilineatus and X. pygmaeus. The DAB locus showed higher levels of genetic variation in the B1-encoding region, (putative binding region) than the DXB locus. We used two methods to investigate d(N)/d(S) ratios. The results from a maximum likelihood method based on phylogenetic relationships indicated positive selection on the B1 region of DAB (this method could not be used on DXB). Results from a coalescent-based method also showed evidence for positive selection in the B1 region of DAB, but only weak evidence for selection on the DXB. Further analyses indicated that recombination is an important source of variation in the B1 region of DAB, but has a relatively small effect on DXB. Overall, our results were consistent with the hypothesis that the DAB locus is under positive selection driven by antagonistic coevolution, and that the DXB locus plays the role of a non-classical MHC II locus. We also used simulations to investigate the presence of an elevated synonymous substitution rate in the binding region. The simulations revealed that the elevated rate could be caused by an interaction between positive selection and codon bias.

Characterization of anti-channel catfish MHC class IIbeta monoclonal antibodies.

This study characterizes four monoclonal antibodies (mAb) developed against the major histocompatibility complex (MHC) class II beta chain of the channel catfish, Ictalurus punctatus. Immunoprecipitations using catfish clonal B cells revealed that each of these mAbs immunoselected proteins of approximately 32 and 36 kD, which are of the appropriate sizes for MHC class II alpha and beta chains, respectively. Cell distribution studies using a fluorescence-activated cell sorter (FACS) combined with RT-PCR analyses demonstrated that MHC class II beta is expressed at a high density on catfish clonal macrophage, B and T cell lines, on alloantigen stimulated leukocytes, and on lipopolysaccharide-induced B-cell blasts. Collectively, these results demonstrate the potential importance of these antibodies as reagents in future studies dealing with the functional role of MHC class II molecules in immune recognition of self from non-self.

Angle-resolved Mueller matrix study of light scattering by B-cells at three wavelengths of 442, 633, and 850 nm.

Angle-resolved signals of polarized light scattered by biological cells provide rich information on cell morphology. Quantitative study of these signals can lead to new methods to develop and improve high-throughput instrumentation for cell probing such as scattering-based flow cytometry. We employ a goniometer system with a photoelastic modulation scheme to determine selected Mueller matrix elements of B-cell hydrosol samples. The angular dependence of S(11), S(12), and S(34) is determined from the scattered light signals between 10 and 160 deg at the three wavelengths 442, 633, and 850 nm. A finite-difference, time-domain (FDTD) method and coated-sphere model are used to investigate the effect of nuclear refractive index on the angle-resolved Mueller elements at different wavelengths using the 3-D structures of selected B cells reconstructed from confocal images. With these results, we demonstrate the value of the light-scattering method in obtaining the cell morphology information.

Chromosomal analysis and identification based on optical tweezers and Raman spectroscopy: reply.

We appreciate the authors' comments in their reply: "On the identification of chromosomes with Raman spectroscopy: a critical comment" [Opt. Express 15, 5997 (2007)]. Their main concern with our paper is asking if the collected spectra have shown the identification or differentiation between three human chromosomes. We think this comment is flawed because the authors misunderstood the main points of the original paper and interpreted the presented spectra data (Fig. 3 and Table 1) incorrectly.

Characterization of glycans on major histocompatibility complex class II molecules in channel catfish, Ictalurus punctatus.

The glycans associated with mammalian major histocompatibility complex (MHC) class II molecules have been studied extensively. Co-translational and post-translational addition of sugar molecules to proteins confers many structural and modulatory functions. In the present study we characterized the glycans associated with MHC class II molecules in the channel catfish to compare glycosylation patterns in a teleost to those known to occur in mammals. This study made use of enzymatic methods and two-dimensional (2D) gel electrophoresis to characterize the N-linked sugars. Unlike mammalian T cells which expressed complex N-linked sugars, channel catfish derived 28S T cells were found to express high-mannose/hybrid N-glycans on class II molecules. However studies with Endoglycosidase H in conjunction with cell surface labeling on peripheral blood leukocytes revealed that catfish possess the machinery to modify the intermediate high-mannose sugars to complex type sugars. Nonetheless, the majority of the class II cell surface glycoproteins were of the high-mannose type. Resolution of catfish MHC class II molecules by 2D gel analyses revealed multiple bands for class II beta chains whereas class II alpha chains focused as a single spot. Glycosylation in the channel catfish, a premier model system for studying the immune system of teleosts, has significant differences from the glycosylation patterns characterized in mammalian systems, likely with functional implications.

Chromosomal analysis and identification based on optical tweezers and Raman spectroscopy.

The ability to identify specific chromosomes with certainty has been established by the development of several cytogenetic techniques based on staining. Here, we report the use of a new optical technique, laser tweezers and Raman spectroscopy (LTRS), to capture and manipulate chromosomes in order to obtain their spectral patterns for molecular analysis without the need for staining. The purpose of this study was to obtain Raman spectroscopy patterns for chromosomes number 1, 2, and 3 and to test if the Raman spectroscopy pattern could be used to distinguish these three chromosomes. In our experiment, optical tweezers were used to capture the individual chromosomes and the Raman spectral patterns were collected for the trapped chromosomes. Then, the captured chromosome was manipulated with the optical tweezers and moved to another chamber through a micro - channel, in which the chromosomes were G- banded for positive identification as chromosome number 1, 2, or 3. Generalized discriminate analysis (GDA) was used to compare the Raman signatures. This analysis revealed that chromosomes 1, 2, and 3 could be distinguished and identified based on their Raman spectra. Development of this approach will lead to more rapid automatic methods for chromosome analysis and identification without the use of prior staining. Moreover, the Raman spectral patterns may lend themselves to more detailed analysis of chromosomal structure than is currently available with standard staining protocols. Such analysis may some day be useful for rapid, automated screening and diagnosis for certain cancers.

Activation-dependent phases of T cells distinguished by use of optical tweezers and near infrared Raman spectroscopy.

Near-infrared Raman spectroscopy may provide a highly sensitive, noninvasive means to identify activation status of leukocytes. The purpose of the current study was to establish Raman spectroscopic characteristics of T cell activation. Activation of the RsL.11 T cell clone in vitro with Con A resulted in specific decrements in band intensities at 785, 1048, 1093, and 1376 cm(-1) but did not alter a majority of other band intensities including those at 1004 cm(-1) (phenylalanine) and 1660 cm(-1) (amide bonds). Activation-dependent decrements in these band intensities occurred subsequent to IL-2 production and correlated closely with T cell blastogenesis. Activation-dependent decrements in these band intensities were not strictly a function of cell size because the same observations were noted in size-controlled comparisons of resting and activated T cells. Like the RsL.11 clone, freshly isolated thymocytes that were activated by Con A or IL-2 showed decrements in particular emissions. These findings indicate that near-infrared Raman spectroscopy can be used as a noninvasive technique to reveal the activation status of single living T cells.

Alternative splicing of major histocompatibility complex class II DXB transcripts in Xiphophorus fishes.

Classical MHC class II glycoproteins present peptides to T cells. In Xiphophorus fishes and in the guppy, Poecilia reticulata, a classical MHC class II B-like transcript has been identified, DAB, as well as a divergent MHC class II B-like transcript, DXB. In the two species of Xiphophorus fishes studied here, X. multilineatus and X. pygmaeus, alternative splicing of the DXB transcript was observed, but not of the classical type DAB transcripts. Two alternative splice patterns were found: a 16-codon deletion and a five-nucleotide deletion that leads to an extension of the transcript. A single DXB transcript that terminates before the transmembrane region was also observed. The alternative splice pattern and the divergence of DXB from DAB suggest that in fish, DXB may have an alternate function. Alternative splicing transcripts of DXB may allow for signaling and localization of DXB within the cell.

Characterization of the molecular chaperone calnexin in the channel catfish, Ictalurus punctatus, and its association with MHC class II molecules.

Folding and assembly of MHC molecules in mammals occurs in the endoplasmic reticulum (ER), but has not been studied in teleosts. Calnexin (CNX) is an ER chaperone that associates with glycoproteins bearing a monoglucosylated N-linked oligosaccharide side chain. Here we report the first identification and characterization of a full-length CNX cDNA clone in a teleost, and the association of the CNX chaperone with MHC class II in a channel catfish T cell line. The 1.8 kb CNX clone encodes a protein of 607 amino acids that is 72% identical to the consensus sequence of mammalian CNXs. The association of CNX with class II is of particular interest because the native MHC class II alpha chain of Ictalurus punctatus does not bear any N-linked oligosaccharide consensus glycosylation sequences. Thus the assembly of class II molecules in the catfish probably proceeds via different steps than occurs in mammals.

IL-4 responsive CD4+ T cells specific for myelin basic protein: IL-2 confers a prolonged postactivation refractory phase.

This study compared myelin basic protein-specific T cells from Lewis rats that were derived in the presence of either rat IL-4 or IL-2. Interleukin-4 was a maintenance factor that enabled derivation of long-term T cell lines. When activated, IL-4 dependent lines were lacking in IL-2 production capacity but maintained high levels of responsiveness to IL-2 and recognized IL-2 as a dominant growth factor. Activated IL-4 dependent T cells rapidly reverted to a quiescent phenotype in the presence of IL-4 and rapidly regained myelin basic protein reactivity. In contrast, activated IL-2 dependent T cells that were propagated in IL-2 had a more persistent blastogenic phenotype and a prolonged refractory phase. Interleukin-4 dependent lines that were propagated in IL-2 up-regulated the capacity to produce IL-2 and also acquired prolonged postactivation refractoriness. Thus, IL-2 was a dominant growth factor that conferred prolonged activation-dependent non-responsiveness. The coupling of dominant growth factor activity with prolonged postactivation refractoriness may be associated with the requisite role of IL-2 in homeostatic self-tolerance.