Genomic sequence, organization, and chromosomal localization of human JAK3.

Members of the Janus (JAK) protein tyrosine kinase family including JAK3 have recently emerged as important components in cytokine signal transduction. Mutations of JAK3 have been found in a number of patients who present with severe combined immunodeficiency. To facilitate the further identification of JAK3-SCID patients and to understand the structure of JAK3 better, we undertook the determination of the genomic sequence, organization, and chromosomal localization of the JAK3 gene. The JAK3 gene was found to consist of 19 exons and 18 introns. Interestingly, the organization of the kinase-(JH1) and pseudokinase-(JH2) domains were found to be dissimilar. In addition, the JAK3 gene was localized to human chromosome 19p13.1. These data should facilitate the identification of patients with this new form of immunodeficiency and will provide insight into the structure of this kinase.

Effective peritoneal and fascial closure of abdominal trocar sites utilizing the Endo-Judge.

With the increasing frequency of minimally invasive surgical procedures, we have begun to see descriptions of new and unforseen complications. One such complication is the formation of a ventral hernia through an unclosed or poorly closed fascial defect created by trocar insertion. The necessity to perform fascial closure of trocar insertion sites, particularly those greater than 5 mm, has been established and is routinely practiced by the majority of laparoscopists. Standard suture techniques can be difficult and frustrating, and often involve blind closure of the fascial defect. A number of instruments have been developed to facilitate this fascial closure. We are currently using a self-contained disposable fascial closure device (Endo-JudgeTM--Synergistic Medical Technologies, Inc., Orlando, Florida), which is quick and relatively simple to use. It enables secure fascial closure under direct vision with the pneumoperitoneum intact. Initial results reveal consistent fascial and peritoneal closure and no postoperative hernia formation.

Identification of the chimeric protein product of the CBFB-MYH11 fusion gene in inv(16) leukemia cells.

An expressed gene formed by fusion between the CBFB transcription factor gene and the smooth muscle myosin heavy chain gene MYH11 is consistently detected by reverse transcription polymerase chain reaction (RT-PCR) in patients who have acute myeloid leukemia (AML) subtype M4Eo with an inversion of chromosome 16. We have previously shown that a CBFB-MYH11 cDNA construct can produce a chimeric protein and transform NIH 3T3 cells. However, the presence of the chimeric protein in patient cells has not been demonstrated previously. Here, we show that such chimeric proteins can be identified in vivo, primarily in the nuclei of the leukemic cells, by use of antibodies against the C-terminus of the smooth muscle myosin heavy chain and the fusion junction peptide. A very high molecular weight protein/DNA complex is generated when nuclear extracts from patient cells are used in electrophoretic mobility shift assays, as seen in NIH 3T3 cells transfected with the CBFB-MYH11 cDNA. Immunofluorescence staining shows that the proteins are organized in vivo into novel structures within cell nuclei. One isoform of the transcript of the CBFB-MYH11 fusion gene, containing the MHC204 C-terminus, was the predominant from in all five cases studied.

Differential basal protein tyrosine phosphorylation in natural killer (NK) and T cells: a biochemical correlate of lymphoid functional activity.

Despite the similarities between natural killer (NK) and T cells, these lymphocytes have dramatically different functional phenotypes. To identify potential biochemical parameters that correlate with the "primed" NK phenotype, we have investigated protein tyrosine phosphorylation in NK and T cells. Examination of tyrosyl phosphorylation in NK cells showed that they have higher levels of phosphorylation than resting T cells. Consistent with this, the concentrations of the tyrosine kinase inhibitor, herbimycin A, required to inhibit FcR-mediated Ca2+ flux in NK cells were much higher than those required for inhibition of T cell receptor-mediated Ca2+ mobilization. Differences in phosphorylation were not due to purification artifact lymphocyte src-family kinase, p56lck or the protein tyrosine phosphatase CD45. Thus, we have identified high basal tyrosyl phosphorylation as a striking biochemical feature of NK cells that correlates with the unique functions of this subset.

Phosphorylation and activation of the Jak-3 Janus kinase in response to interleukin-2.

Interleukin-2 is an autocrine growth factor for T cells which also activates other cells including B cells and natural killer cells. The subunits of the interleukin-2 receptor (IL-2R) lack intrinsic enzymatic activity, but protein tyrosine phosphorylation is a critical event following ligand binding and src family kinases, such as Lck, are known to be activated by IL-2 (refs 5-9). However, IL-2 signalling can occur in the absence of receptor interaction with Lck, suggesting that other protein tyrosine kinases might be important. Here we report that a new member of the Janus family of kinases (Jak-3) is coupled to the IL-2R in human peripheral blood T cells and natural killer cells.

Molecular cloning of L-JAK, a Janus family protein-tyrosine kinase expressed in natural killer cells and activated leukocytes.

Protein-tyrosine kinases (PTKs) are critical enzymes for receptor-mediated signaling in lymphocytes. Because natural killer (NK) cells are large granular lymphocytes with specialized effector function, we set out to identify PTKs preferentially expressed in these cells. One such PTK was identified and molecularly cloned. The predicted amino acid sequence shows that this kinase lacks SH2 or SH3 domains typical of src family kinases but has tandem nonidentical catalytic domains, indicating that it is a member of the Janus family of PTKs. Immunoprecipitation using antiserum generated against a peptide corresponding to the deduced amino acid sequence of this gene revealed a kinase with a molecular weight of approximately 125,000. The pattern of expression of this kinase contrasted sharply with that of other Janus kinases, which are ubiquitously expressed. The kinase described in the present study was found to be more limited in its expression; expression was found in NK cells and an NK-like cell line but not in resting T cells or in other tissues. In contrast, stimulated and transformed T cells expressed the gene, suggesting a role in lymphoid activation. Because of its homology and tissue expression, we have tentatively termed this PTK gene L-JAK for leukocyte Janus kinase.