Several functional anomalies of B-chronic lymphocytic leukemia (B-CLL) cells may be explained by abnormalities of the B-cell receptor (BCR), a multimeric complex formed by the sIg homodimer and the noncovalently bound heterodimer Ig/Igβ (CD79a/CD79b). Because the expression of the extracellular Ig-like domain of CD79b has been reported to be absent in the cells of most CLL cases, we have investigated the molecular mechanisms that may account for this defect. Peripheral blood lymphocytes (PBL) from 50 patients and two cell lines (MEC1, MEC2) obtained from the PBL of one of them were studied. MEC1, MEC2, and 75% of CLL cases did not express detectable levels of the extracellular Ig-like domain of CD79b, which was nevertheless present in greater than 80% CD19+ cells from normal donors. In healthy subjects the expression of CD79b was equally distributed in CD5+ and CD5− B-cell subsets. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of CD79b RNA from all patients and from MEC1 and MEC2 cell lines consistently yielded two fragments of different size (709 bp and 397 bp). The 709-bp band corresponds to CD79b entire transcript; the 397-bp band corresponds to an alternatively spliced form lacking exon 3 that encodes the extracellular Ig-like domain. Both fragments were also visible in normal PBL. The expression of the 397-bp fragment was increased in normal activated B cells, while no difference was seen between CD5+ and CD5− B cells. To obtain a more accurate estimate of the relative proportions of the two spliced forms, a radioactive PCR was performed in 13 normal and 22 B-CLL samples and the results analyzed using a digital imager. The mean value of the CD79b to the CD79b internally deleted ratio was 0.64 ± 0.20 SD in normal donors and 0.44 ± 0.27 SD in B-CLL (P = .01). Direct sequencing of 397-bp RT-PCR products and of genomic DNA corresponding to exon 3 from MEC1, MEC2, their parental cells, and five fresh B-CLL samples did not show any causal mutation. Single-strand conformation polymorphism analysis of exon 3 performed in 18 additional B-CLL cases showed a single abnormal shift corresponding to a TGT → TGC polymorphic change at amino acid 122. We propose a role for the alternative splicing of CD79b gene in causing the reduced expression of BCR on the surface of B-CLL cells. As normal B cells also present this variant, the mechanism of CD79b posttranscriptional regulation might reflect the activation stage of the normal B cell from which B-CLL derives.

In mammalian cells, many of the enzymes required for the production of dNTPs are regulated during the cell cycle such that their activities increase during S phase, the time of DNA replication (1). In some instances the molecular mechanisms creating this phenomenon are known in detail and affect both transcription and translation (2-7). The magnitude of the effect varies greatly for different enzymes. Thymidine kinase (2-4) and ribonucleotide reductase (5-7) show low activity outside S phase, with large increases during S phase. Conversely, deoxycytidine kinase (8) is constitutively active, and also thymidylate synthase undergoes only minor variations during the cell cycle (9). It is noteworthy that the activity of enzymes belonging to the de novo pathway as well as enzymes of the salvage pathway may either vary greatly or not at all during the cell cycle.This picture comes largely from experiments in which the activity of enzymes was measured either directly in cell extracts or by an in situ assay in intact cells. The latter method is of greater physiological relevance. It involves incubation of cells in culture with highly radioactive ribo-or deoxyribonucleosides, usually followed by an analysis of the time-dependent flow of isotope through the nucleotide pools into nucleic acids and, in appropriate cases, into excretion products in the medium. Inhibitors (9 -12) or mutations (13, 14) affecting enzymes of dNTP synthesis influence the flow of isotope and can give information about the normal process. Two major pitfalls of this technology are all too often neglected (1): (i) heterogeneity in the cell population under study and (ii) changes in the specific radioactivity of the dNTP pools. The two factors may combine and distort the result to a point where they are misinterpreted (14). This was first recognized in experiments with Chinese hamster ovary cells (15) and further studied in more detail with V79 (16) and CEM (13) cells. Consider a population of cycling cells in culture in which the DNA is labeled from a nucleoside. Fig. 1 shows the series of reactions that, after the entry of a labeled nucleoside into the cell, transform the nucleoside into a labeled dNTP prior to incorporation into DNA. A deoxyribonucleoside, such as thymidine or deoxycytidine, is transformed directly via three phosphorylation steps, whereas a ribonucleoside, such as cytidine, is first phosphorylated to CDP, followed by reduction to dCDP by ribonucleotide reductase, and finally phosphorylated to dCTP. The radioactive nucleotides mix with nucleotides synthesized de novo, resulting in dilution of radioactivity. The specific radioactivity of a dNTP pool thus depends not only on the efficiency with which the nucleoside is transformed to the dNTP but also on the rate at which the dNTP is synthesized de novo. An additional important consideration is the rate with which the dNTP is removed, either by its catabolism or by its utilization for DNA synthesis. The specific activity of the dNTP gives an indication of the relative activities of all ...

After phosphorylation to the corresponding diphosphates, 2'-azido-2'-deoxycytidine and 2'-difluorocytidine act as powerful inhibitors of ribonudeotide reductase.

The B cell antigen receptor (BCR) includes an Igα/Igβ heterodimer non‐covalently associated with surface immunoglobulin. Recently, variant Igα and Igβ transcripts, arising fromalternative mRNA splicing, have been reported. The present study examined the function of the potential products of these transcripts, by utilizing cDNA expression plasmids to reconstitute human BCR expression in transfected 293T cells. Spliced transcripts produced truncated proteins (ΔIgα and ΔIgβ), that failed to form heterodimers with their full‐length counterparts, and didnot mediate transport of IgM to the cell surface. When overexpressed, both ΔIgα and ΔIgβ acted as competitors of Igα and Igβ, leading to down‐modulated surface IgM expression, and retention of IgM in the endoplasmic reticulum. These findings document a possible novel mechanism for controlling BCR expression in B cells, based on up‐regulated synthesis of components devoid of transport function.

As thymocyte infection may represent one of the mechanisms responsible for CD4+ T lymphocyte depletion in HIV-1-infected individuals, we studied the occurrence of HIV-1 infection in the thymus in vivo. Thymus (THYPD) and peripheral blood (PBLPD) primary viral isolates were obtained from an HIV-1-infected patient; restriction pattern analysis revealed the presence of a viral variant (THY) in the thymus isolate, from which biological viral clones containing this variant were obtained by limiting dilution infection of Molt-3 cells. The biological phenotype of the viral isolates and THY clones was studied in different cell lines and primary cultures. PBLPD, THYPD, and THY clones could efficiently infect T cell lines; the thymic variant showed a higher cytopathic activity in T cell lines, and a higher replication capacity in both unfractionated and CD4+CD8(+)-enriched primary thymocytes. Sequence analysis of the viral population patterns in vivo confirmed the presence of the THY variant in the thymic compartment, and revealed that the degree of V3 loop heterogeneity was higher in the thymocytes of the patient than in the peripheral blood lymphocytes. In addition to confirming thymocyte infection in vivo, our data also indicate that a differential distribution of viral variants may occur among different body compartments in a single individual; the emergence of cytopathic and tissue-specific variants in the thymus may play a relevant role in the pathogenesis of HIV-1 disease.

Conventional karyotypes, NOR-bearing chromosomes by means of silver impregnation and genome size were investigated in five Mediterranean species in three genera of the Syngnathidae. A karyotype of 48 subtelocentric-acrocentric chromosomes was found in the seahorse Hippocampus hippocampus (FN=48) while a diploid value of 44 occurred in H. guttulatus (2 sm-m+42 a; FN=46) and the pipefish Syngnathus abaster (44 a; FN=44) and S. typhle (44 a; FN=44). The pipefish Nerophis ophidion, possessing a diploid chromosomal set of 58 made up of 50 meta-submetacentric and eight subteloacrocentric elements (FN=108) and a genome size three to four times larger than those known to date, differs cytogenetically from all other Syngnathids studied so far. A single pair of active NOR-bearing chromosomes was found in both species of the genus Hippocampus while in Syngnathus and Nerophis species more than two silver positive chromosomes were found to be involved in nucleolus organization giving rise to NOR-bearing chromosome polymorphism. The possible evolutionary routes of quantitative and qualitative changes in chromosome and DNA are discussed. The resulting phylogenetic scheme is shown to coincide with that constructed from morphological characters. 1998 The Fisheries Society of the British Isles

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In view of our recent findings that a truncated form of the CD4 and sera from SIV-infected macaques. We also envelope (Env) glycoprotein of human immunodeficiency observed pseudotype-mediated gene transfer of a green virus type 1 (HIV-1) was efficiently incorporated into fluorescent protein marker into the CD4 + CEMX174 and MoMLV particles, we studied the generation of Moloney C8166 lymphoid cell lines. More importantly, primary murine leukemia virus (MoMLV)/simian immunodeficiency human lymphocytes were also successfully transduced ex virus (SIV) pseudotypes. Unlike HIV-1, both the wild-type vivo by MoMLV/SIV pseudotypes, albeit at lower efficiency, SIV Env and a truncated form, which lacks most of the and gene transfer was specifically restricted to the CD4 + cytoplasmic domain of the transmembrane glycoprotein, subset. These findings demonstrate that MoMLV/SIV were incorporated into MoMLV particles and generated pseudotypes can be used to transduce cells which are susinfectious retroviral vectors which could transduce CD4 + ceptible to SIV infection, and thus might be advantageously sMAGI macaque cells. The infection depended on target employed in animal models for direct in vivo delivery of cell CD4 expression, and was neutralized by both soluble gene therapy-based approaches.