Arno Helmberg

CYP11B1 mutations causing congenital adrenal hyperplasia due to 11 beta-hydroxylase deficiency.

Accurate knowledge of the molecular basis of congenital adrenal hyperplasia due to 11 beta-hydroxylase deficiency is a prerequisite for genetic counseling, prenatal diagnosis, and treatment. Analysis of nine patients suffering from severe manifestations of this disorder led to the identification of seven novel mutations in their CYP11B1 genes. A Caucasian patient was homozygous for the missense mutation R448H, previously found only in Jews of Moroccan origin. An Iranian patient was found to be homozygous for a different mutation in the same codon, R448C. Of four unrelated patients, two were homozygous for a nonsense mutation (W247X), whereas two others were compound heterozygotes for W247X in combination with either R448H or E371G. Two other patients were homozygous for either the missense mutation A331V or an in-frame CTG insertion adjacent to codon 464 (InsCTG464). One patient was a compound heterozygote for two mutations in exon 2, a 28-bp deletion (delta 28bpEx2) and the missense mutation V129M. All of the missense mutations and the CTG insertion caused a complete loss of steroid 11 beta-hydroxylating activity when expressed in cultured cells. These data support previous suggestions of mutational hot spots in CYP11B1 and confirm that severe clinical manifestations are associated with complete loss of enzymatic activity.

Gene expression profiles of proliferating vs. G1/G0 arrested human leukemia cells suggest a mechanism for glucocorticoid-induced apoptosis.

Glucocorticoids (GC) have pronounced effects on metabolism, differentiation, proliferation, and cell survival (1). In certain lymphocytes and lymphocyte‐related malignancies, GC inhibit proliferation and induce apoptotic cell death, which has led to their extensive use in the therapy of malignant lymphoproliferative disorders (2). Most of these effects result from regulation of gene expression via the GC receptor (GR), a ligand‐activated transcription factor (3). Although hundreds of genes are regulated by GC (1), how certain biological GC effects relate to individual gene regulation remains enigmatic. To address this question with respect to GC‐induced cell cycle arrest and apoptosis, we applied DNA chip technol¬ogy (4, 5) to determine gene expression profiles in proliferating and G1/G0‐arrested (by conditional expres¬sion of the CDK inhibitor p16/INK4a) acute lymphoblas¬tic Τ cells undergoing GC‐induced apoptosis. Of 7074 genes tested, 163 were found to be regulated by dexamethasone in the first 8 h in proliferating cells and 66 genes in G1/G0‐arrested cells. An almost nonoverlapping set of genes (i.e., only eight genes) was coordinately regulated in proliferating and arrested cells. Analysis of the regulated genes supports the concept that GC‐induced apoptosis results from positive GR autoregulation entail¬ing persistent down‐regulation of metabolic pathways crit¬ical for survival.—Tonko, M., Ausserlechner, M. J., Bernhard, D., Helmberg, A., Kofler, R. Gene expression profiles of proliferating vs. G1/G0 arrested human leu¬kemia cells suggest a mechanism for glucocorticoid‐induced apoptosis. FASEB J. 15, 693‐699 (2001)

Mouse Vk gene classification by nucleic acid sequence similarity

Analyses of immunoglobulin (Ig) variable (V) region gene usage in the immune response, estimates ofV gene germline complexity, and other nucleic acid hybridization-based studies depend on the extent to which such genes are related (i. e., sequence similarity) and their organization in gene families. While mouseIgh heavy chainV region (VH) gene families are relatively well-established, a corresponding systematic classification ofIgk light chainV region (Vk) genes has not been reported. The present analysis, in the course of which we reviewed the known extent of theVk germline gene repertoire andVk gene usage in a variety of responses to foreign and self antigens, provides a classification of mouseVk genes in gene families composed of members with >80% overall nucleic acid sequence similarity. This classification differed in several aspects from that ofVH genes: only someVk gene families were as clearly separated (by >25% sequence dissimilarity) as typicalVH gene families; mostVk gene families were closely related and, in several instances, members from different families were very similar (>80%) over large sequence portions; frequently, classification by nucleic acid sequence similarity diverged from existing classifications based on amino-terminal protein sequence similarity. Our data have implications forVk gene analyses by nucleic acid hybridization and describe potentially important differences in sequence organization betweenVH andVk genes.

Mouse Variable-Region Gene Families: Complexity, Polymorphism and Use in non-Autoimmune Responses

During B-cell ontogeny, functional immunoglobulin (Ig) genes are generated by somatic juxtaposition of gene segments separated in the germline and termed variable (V), diversity (D, heavy chain only) and joining (J) gene segments (reviewed in (Tonegawa 1983, Alt et al. 1986)). V genes encode all residues of the first and second complementarity-determining region (CDR) of both heavy (H) and light (L) chains as well as part of the L chain CDR-3, and hence contribute the majority of antigen contact residues (Kabat et al. 1991). In human and murine systems, a considerable number of highly polymorphic VH and VL gene segments exist in the germline and, for practical reasons, have been classified into V-gene families and V-region protein groups. The complexity of V-gene families, their chromosomal organization and degree of polymorphism, and their usage in various responses has been studied in great detail. In an attempt to better understand autoimmune diseases, the present volume addresses the expression of these V genes and V-gene families in response to self antigens. This chapter introduces Ig genetics in the mouse, summarizes current Vgene and V-region protein classifications, reviews the complexity, chromosomal organization and polymorphism of the V-gene repertoire, and discusses V-gene usage in different B-cell repertoires and in response to complex foreign antigens to allow comparisons with V-gene usage in autoantibodies described throughout this volume. Lambda L chains are not included since they make up only a few percent of total mouse serum Ig and are rarely found in autoantibodies. Furthermore, the large body of data on V-gene usage in hapten responses will

Expression profiling of glucocorticoid-treated T-ALL cell lines: rapid repression of multiple genes involved in RNA-, protein- and nucleotide synthesis

To arrive at a better understanding of the effects of the glucocorticoid component of chemotherap#y protocols on lymphocytic leukemia cells, we analysed early responses of T-lymphocytic leukemia cell lines Jurkat and CEM-C7, both of which undergo apoptosis in response to dexamethasone, via gene chips. Among genes identified as repressed, a notable cluster seemed to be of importance for the processes of transcription, mRNA splicing and protein synthesis. Consequently, we assessed time-resolved uptake of uridine and methionine to monitor RNA and protein synthesis, along with parameters quantifying apoptosis. Repression of uptake to about 65% of that in untreated cells preceded the first sign of apoptosis by several hours in both cell lines. In addition to this general repression of RNA and protein synthesis, several genes were found to be regulated that may contribute to synergistic action of glucocorticoids with other components of frequently used chemotherapy protocols such as antimetabolites, methotrexate and alkylating agents.

c-Myc does not prevent glucocorticoid-induced apoptosis of human leukemic lymphoblasts

Due to their growth arrest- and apoptosis-inducing ability, glucocorticoids (GC) are widely used in the therapy of various lymphoid malignancies. The signal transduction pathways leading to this clinically-relevant form of apoptosis have, however, not been sufficiently elucidated. GC bind to their specific receptor, a ligand-activated transcription factor of the Zn-finger type, that activates or represses transcription of GC-responsive genes. Previous studies in leukemia cells suggested that transcriptional repression of c-myc expression might be the crucial event in GC-induced apoptosis, although in other systems, c-Myc apparently increased the sensitivity to cell-death inducers. To address this controversy, we stably transfected the GC-sensitive human T-ALL cell line CEM-C7H2 with constructs allowing tetracycline-regulated expression of c-Myc. Subsequent analyses of these cell lines showed that overexpression of c-Myc per se had little, if any, effect on cell viability, although it rendered the cells more sensitive to apoptosis induced by low serum, confirming the functionality of the expressed transgene. More importantly, however, when the cells were treated with GC in the presence of exogenous c-Myc, they underwent apoptosis exceeding that in cells treated in the absence of transgenic c-Myc. The data indicate that c-myc downregulation is not critical for induction of cell-death by GC in this system, and support the notion that c-Myc sensitizes cells to apoptosis-inducing agents.

DNA-microarrays: novel techniques to study aging and guide gerontologic medicine

DNA array technology is a high throughput application of nucleic acid hybridization. Arrays currently used include spotted cDNA arrays at various densities and oligonucleotide arrays, where the highest densities can be obtained by synthesizing oligonucleotides directly on the solid support. While cDNA arrays can only determine the presence of a specific sequence in a sample, oligonucleotide arrays additionally allow detection of mutations and polymorphisms, including single nucleotide polymorphisms. Microarray technology can be expected to further our understanding of the aging process and to soon influence medical decision-making in age-related diseases, as well as lifestyle decisions of consequence to the aging process.

Mechanism of allergic cross-reactions--III. cDNA cloning and variable-region sequence analysis of two IgE antibodies specific for trinitrophenyl.

As a first step toward defining the molecular interactions between ligands and the IgE antigen-combining site, we report here the cDNA cloning and variable (V) region nucleic acid sequences of the heavy (H) and light (L) chains of 2 monoclonal mouse IgE antibodies to trinitrophenyl (ATCC-TIB142 = IGELa2 and ATCC-TIB141 = IGELb4). In all instances, full-length cDNA clones were obtained to facilitate future expression studies. The H chains were encoded by VH genes from the VH3660 and J558 gene families in context with DQ52 and DSP2.2 diversity (D) mini genes, and JH3 and JH4 joining (J) gene segments, respectively. Vk8/Jk2 and Vk1/Jk5 rearrangements encoded the respective L chain V-regions. Both antibodies exhibited considerable conservation of complementarity determining region (CDR) sequences, which will facilitate template-based computer modeling of the three-dimensional structures of complexes formed between various ligands and these antibodies. From sequence comparison between the dinitrophenyl (DNP)-binding myeloma protein MOPC-315 and these IgE antibodies likely candidates for hapten-contact residues within the binding sites of IGELa2 and IGELb4 have been suggested.

Evolutionary relationship between human and mouse immunoglobulin kappa light chain variable region genes

Similar to the Igh-V multigene family, the human or mouse Igk-V repertoirer is a distorted continuum of homologous genes that may be grouped into families displaying >80% nucleic acid sequence similarity among their members. systematic interspecies sequence comparisons reveal that most human Igk-V gene families exhibit clear homology to mouse Ogk-V families (sequence similarity >74%). A hypothetical phylogenetic tree of Igk-V genes predicts that a minimum of seven Igk-V genes/families predate mammalian radiation. In two cases, several interrelated mouse Igk-V families exhibit phylogenetic equidistance with just one human Igk-V family, implying a more pronounced divergence for the elevated number of Igk-V gene families in the mouse. Mouse-human Igk-V comaprisons, moreover, illustrate how expansion, contraction, and perhaps deletion of Igk-V gene families shape the Igk-V repertoire during mammalian evolution.