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L J Kienker

Publications and source records attributed to L J Kienker.

10 recordsLinked to original sources
PubMed2000

Both V(D)J recombination and radioresistance require DNA-PK kinase activity, though minimal levels suffice for V(D)J recombination.

DNA-dependent protein kinase (DNA-PK) is utilized in both DNA double-strand break repair (DSBR) and V(D)J recombination, but the mechanism by which this multiprotein complex participates in these processes is unknown. To evaluate the importance of DNA-PK-mediated protein phosphorylation in DSBR and V(D)J recombination, we assessed the effects of the phosphatidyl inositol 3-kinase inhibitor wortmannin on the repair of ionizing radiation-induced DNA double-strand breaks and V(D)J recombination in the V(D)J recombinase inducible B cell line HDR37. Wortmannin radiosensitized HDR37, but had no affect on V(D)J recombination despite a marked reduction in DNA-PK activity. On the other hand, studies with mammalian expression vectors for wild-type human DNA-PK catalytic subunit (DNA-PKcs) and a kinase domain mutant demonstrated that only the kinase active form of DNA-PKcs can reconstitute DSBR and V(D)J recombination in a DNA-PKcs-deficient cell line (Sf19), implying that DNA-PKcs kinase activity is essential for both DSBR and V(D)J recombination. These apparently contradictory results were reconciled by analyses of cell lines varying in their expression of recombinant wild-type human DNA-PKcs. These studies establish that minimal DNA-PKcs protein levels are sufficient to support V(D)J recombination, but insufficient to confer resistance to ionizing radiation.

Androstadienes↗
PubMed1998

Regulatory elements in the promoter of a murine TCRD V gene segment.

TCRD V segments rearrange in an ordered fashion during human and murine thymic development. Recombination requires the accessibility of substrate gene segments, and transcriptional enhancers and promoters have been shown to regulate the accessible chromatin configuration. We therefore investigated the regulation of TCRD V rearrangements by characterizing the promoter of the first TCRD V segment to be rearranged, DV101S1, under the influence of its own enhancer. Sequences required for full promoter activity were identified by transient transfections of normal and mutated promoters into a human gammadelta lymphoma, and necessary elements fall between -86 and +66 nt, relative to the major transcription start site. They include a cAMP responsive element (CRE) at -62, an Ets site at -39, a TATA box at -26, the major transcriptional start site sequence (-8 to -5 and -2 to +11), and a downstream sequence (+12 to +33). Gel shift analyses and in vitro DNase I footprinting showed that nuclear proteins bind to the functionally relevant CRE, Ets, +1 to +10 sequence, and the +17 to +21 sequence. Nuclear proteins also bind to an E box at -52, and GATA-3 binds to a GATA motif at -5, as shown by Ab ablation-supershift experiments, but mutations that abrogated protein binding to these sites failed to affect DV101S1 promoter activity. We conclude that not all protein-binding sites within the DV101S1 minimal promoter are important for enhancer driven TCRD gene transcription. Further, the possibility remains that the GATA and E box sites function in enhancer independent DV101S1 germline transcription.

Animals↗
PubMed1997

Advanced multiplexed analysis with the FlowMetrix system.

The FlowMetrix System is a multiplexed data acquisition and analysis platform for flow cytometric analysis of microsphere-based assays that performs simultaneous measurement of up to 64 different analytes. The system consists of 64 distinct sets of fluorescent microspheres and a standard benchtop flow cytometer interfaced with a personal computer containing a digital signal processing board and Windows95-based software. Individual sets of microspheres can be modified with reactive components such as antigens, antibodies, or oligonucleotides, and then mixed to form a multiplexed assay set. The digital signal-processing hardware and Windows95-based software provide complete control of the flow cytometer and perform real-time data processing, allowing multiple independent reactions to be analyzed simultaneously. The system has been used to perform qualitative and quantitative immunoassays for multiple serum proteins in both capture and competitive inhibition assay formats. The system has also been used to perform DNA sequence analysis by multiplexed competitive hybridization with 16 different sequence-specific oligonucleotide probes.

Alleles↗
PubMed1991

T cell receptor gamma and delta gene rearrangements in scid thymocytes. Similarity to those in normal thymocytes.

The nature of TCR gamma and delta gene rearrangements in 4- to 6-week-old scid thymocytes was examined by using the polymerase chain reaction technique, cloning, and DNA sequencing. Analysis of 78 sequences indicates that TCR gamma and delta gene rearrangements in scid mice generally resemble those in thymocytes from normal young adult mice. V gamma 1, V gamma 2, and V gamma 5 rearrangements are heterogeneous, with extensive N region addition and nucleotide excision from the recombining coding segments. In addition, homogeneous and fetal-like V gamma 3, V gamma 4, and V delta 1 rearrangements are observed. These rearrangements are currently difficult to interpret but may be significant with respect to whether certain homogeneous joints in normal mice are due to cellular selection or to the rearrangement process. scid TCR gamma and delta gene nucleotide sequences also reveal direct V-J delta joining, inter-(V-J-C gamma) cluster joining, and the possibility of inversional rearrangement at the gamma locus. Short sequence homologies may contribute to V(D)J recombination and to the rescue of blocked coding joints.

Animals↗
PubMed1991

T cell receptor gamma and delta gene junctional sequences in SCID mice: excessive P nucleotide insertion.

The severe combined immunodeficiency (SCID) mutation has been postulated to affect a V(D)J recombinase activity involved in coding joint formation. Analysis of 38 joints from 34 distinct sequences of normally rearranged T cell receptor (TCR) gamma and delta genes from adult, SCID thymocytes reveals coding joints with an increased number of P nucleotides. One-third of P sequences are greater than or equal to 4 nucleotides in length and P elements of up to 15 bases are observed. This suggests that the SCID defect deregulates P nucleotide addition. Consequently, essential V(D)J recombination intermediates may seldom be generated.

Animals↗
PubMed1988

Patterns of Ig V region expression among neonatal hemagglutinin-responsive B cells. Evidence for non-random VH gene representation during later stages of development.

Hybridoma libraries were established whose specificities reflect those within the BALB/c hemagglutinin-responsive B cell repertoire at 1 or 2 wk of age. These libraries were generated through chronic immunization regimes that induce responses dominated by clonotypes available at the age of initial immunization. Dot blot analyses of cytoplasmic RNA from these hybridomas were performed to determine the Ig H chain V region (VH) families associated with the repertoire at each age. Although genes from most known VH families can generate hemagglutinin-specific antibodies, clonotypes prevalent during the first week of life disproportionately use VH7183 gene segments. In contrast, hybridomas representative of the repertoire in 2-wk-old individuals preferentially use VHS107, VH36-60, and VHX24 gene segments. These results demonstrate changes in VH gene family predominance that correlate with the age-related patterns of clonal emergence and turnover previously shown in the hemagglutinin-reactive B cell pool. Taken together, these findings suggest that the very early neonatal Ag-responsive B cell pool closely reflects preferential VH gene rearrangements within the pre-B cell compartment. Further, they suggest that either non-random strategies of VH gene expression, or selective clonal expansion strategies based on VH, operate even at later stages of development.

Aging↗
PubMed1981

Inhibition of spontaneous but not antibody-dependent cell-mediated cytotoxicity by simple sugars: evidence that endogenous lectins may mediate spontaneous cell-mediated cytotoxicity.

Using Chang and K-562 cell line cells as targets, we have observed that a number of sugars are capable of inhibiting spontaneous cell-mediated cytotoxicity (SCMC) but not antibody-dependent cell-mediated cytotoxicity (ADCC). The sugars D(-)ribose, beta-gentiobiose, N-acetyl-D-galatosamine, and alpha-lactose all significantly inhibited SCMC of Chang and K-562 cell line cells. Because these same sugars caused no inhibition of ADCC against either Chang or K-562 cell line cells in assays run simultaneously, the results do not appear to be due to a non-specific toxic effect of the sugars against the effector cells. These studies add to the evidence that ADCC and SCMC are mediated by separate receptors. Furthermore, they provide evidence that endogenous lectin receptors or lectin-like molecules may be involved in the recognition and/or effector stages leading to SCMC. Thus, NK cells may recognize targets by virtue of receptors capable of interacting with monosaccharide, disaccharide, or oligosaccharide sequences present alone, as glycolipids, and/or as glycoproteins on the target cell surface.

Antibody-Dependent Cell Cytotoxicity↗