Ultraviolet 365 as an Alternative Light Source for Detection of Blood Serum.

The use of alternative light sources (ALS) in bloodstain analysis has focused on dried (whole) blood, while information on detection of blood serum is lacking. Serum detection by ALS could provide valuable information at a crime scene, as serum may become separated from blood during clotting and cast off, especially in cases where the victim is moved. Additionally, a perpetrator may concentrate on the removal/scouring of dried blood with small amounts of serum going unnoticed, as it dries relatively clear on certain objects. In this report, the detection of human blood serum was evaluated using ultraviolet (UV) light at two different wavelengths. These results show that ultraviolet (UV) at 365 nm (UV365) was effective in the detection of even small amounts of blood plasma and serum, compared with UV at 395 nm, which was not. UV365 was also found to be useful in distinguishing blood imprints from clotting blood which had been transferred to material versus blood that had been added directly. Taken together, these results demonstrate that UV365 may be utilized as a simple, nondestructive method for blood serum detection.

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.

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.

Mutational analysis of conserved amino acids in the T cell receptor alpha-chain transmembrane region: a critical role of leucine 112 and phenylalanine 127 for assembly and surface expression.

Correct assembly of all TCR complex polypeptides is essential for its cell surface expression and function. The transmembrane region of the TCRalpha chain is highly conserved and to gain insight into the structural and functional role of these residues, single amino acid substitutions were introduced and surface expression and signaling ability studied in T hybridoma cells. Introduction of acid residues within the TCRalpha chain transmembrane region were mostly tolerated, indicating that the net charge within this region of the TCR complex is not crucial to either assembly or signaling. However, mutations of leucine 112 or phenylalanine 127 to aspartic acids (L112D or F127D, respectively) resulted in dramatic loss of surface expression and, therefore, their signaling ability. Intracellular flow cytometry showed that the mutant TCRalpha polypeptides were present at levels comparable to wild-type, indicating that the reduced surface expression was not a consequence of impaired protein survival. The defect was characterized by immunoprecipitation and showed that residues L112 and F127 were involved in early interactions with the CD3 complex. A large proportion of the TCRalpha chain mutants L112D and F127D consisted of immature protein, indicative of a problem during early assembly of the TCR. Our findings provide evidence for the involvement of the conserved L112 and F127 residues of the TCRalpha chain transmembrane region in the assembly process of the TCR complex.