Development of a continuous assay for the measurement of tissue factor procoagulant activity on intact cells.

Tissue factor (TF) is the major physiological initiator of the coagulation cascade and has an important function in the morbidity and mortality associated with many disease states, including cancer-associated thrombosis and atherosclerosis. TF normally exists in a partially encrypted state and its de-encryption on circulating monocytes, platelets or endothelial cells by inflammatory mediators can lead to thrombosis. Furthermore, many cancer cells express large amounts of TF and these cells communicate readily with the circulation through the fenestrated tumor endothelium.

Preventing the spontaneous modification of an HLA-A2-restricted peptide at an N-terminal glutamine or an internal cysteine residue enhances peptide antigenicity.

The p68-derived peptide, QIVDVCHDV, was identified by a reverse immunology approach as capable of reconstituting an epitope recognized by the melanoma-reactive cytotoxic T lymphocyte (CTL) line VMM5. The peptide has not been demonstrated definitively on the cell surface by mass spectrometry; thus, it is not yet considered appropriate for use in human melanoma vaccines. Interestingly, however, the antigenicity of this peptide was affected by spontaneous modifications at two distinct residues.

A mitotic cascade of NIMA family kinases. Nercc1/Nek9 activates the Nek6 and Nek7 kinases.

The Nek family of protein kinases in humans is composed of 11 members that share an amino-terminal catalytic domain related to NIMA, an Aspergillus kinase involved in the control of several aspects of mitosis, and divergent carboxyl-terminal tails of varying length. Nek6 (314AA) and Nek7 (303AA), 76% identical, have little noncatalytic sequence but bind to the carboxyl-terminal noncatalytic tail of Nercc1/Nek9, a NIMA family protein kinase that is activated in mitosis. Microinjection of anti-Nercc1 antibodies leads to spindle abnormalities and prometaphase arrest or chromosome missegregation.

Identification of the beta cell antigen targeted by a prevalent population of pathogenic CD8+ T cells in autoimmune diabetes.

Type 1 diabetes is an autoimmune disease in which autoreactive T cells attack and destroy the insulin-producing pancreatic beta cells. CD8+ T cells are essential for this beta cell destruction, yet their specific antigenic targets are largely unknown. Here, we reveal that the autoantigen targeted by a prevalent population of pathogenic CD8+ T cells in nonobese diabetic mice is islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP).

The minor histocompatibility antigen HA-3 arises from differential proteasome-mediated cleavage of the lymphoid blast crisis (Lbc) oncoprotein.

Minor histocompatibility (H) antigens crucially affect the outcome of human leukocyte antigen (HLA)-identical allogeneic stem cell transplantation (SCT). To understand the basis of alloimmune responses against minor H antigens, identification of minor H peptides and their antigenicity-determining mechanisms is essential. Here we report the identification of HA-3 and its encoding gene.

Gene silencing: trans-histone regulatory pathway in chromatin.

The fundamental unit of eukaryotic chromatin, the nucleosome, consists of genomic DNA wrapped around the conserved histone proteins H3, H2B, H2A and H4, all of which are variously modified at their amino- and carboxy-terminal tails to influence the dynamics of chromatin structure and function -- for example, conjugation of histone H2B with ubiquitin controls the outcome of methylation at a specific lysine residue (Lys 4) on histone H3, which regulates gene silencing in the yeast Saccharomyces cerevisiae. Here we show that ubiquitination of H2B is also necessary for the methylation of Lys 79 in H3, the only modification known to occur away from the histone tails, but that not all methylated lysines in H3 are regulated by this 'trans-histone' pathway because the methylation of Lys 36 in H3 is unaffected. Given that gene silencing is regulated by the methylation of Lys 4 and Lys 79 in histone H3, we suggest that H2B ubiquitination acts as a master switch that controls the site-selective histone methylation patterns responsible for this silencing.

Set2 is a nucleosomal histone H3-selective methyltransferase that mediates transcriptional repression.

Recent studies of histone methylation have yielded fundamental new insights pertaining to the role of this modification in gene activation as well as in gene silencing. While a number of methylation sites are known to occur on histones, only limited information exists regarding the relevant enzymes that mediate these methylation events. We thus sought to identify native histone methyltransferase (HMT) activities from Saccharomyces cerevisiae.