In chronic lymphocytic leukemia (CLL) the level of minimal residual disease (MRD) after therapy is an independent predictor of outcome. Given the increasing number of new agents being explored for CLL therapy, using MRD as a surrogate could greatly reduce the time necessary to assess their efficacy. In this European Research Initiative on CLL (ERIC) project we have identified and validated a flow-cytometric approach to reliably quantitate CLL cells to the level of 0.0010% (10−5). The assay comprises a core panel of six markers (i.e. CD19, CD20, CD5, CD43, CD79b and CD81) with a component specification independent of instrument and reagents, which can be locally re-validated using normal peripheral blood. This method is directly comparable to previous ERIC-designed assays and also provides a backbone for investigation of new markers. A parallel analysis of high-throughput sequencing using the ClonoSEQ assay showed good concordance with flow cytometry results at the 0.010% (10−4) level, the MRD threshold defined in the 2008 International Workshop on CLL guidelines, but it also provides good linearity to a detection limit of 1 in a million (10−6). The combination of both technologies would permit a highly sensitive approach to MRD detection while providing a reproducible and broadly accessible method to quantify residual disease and optimize treatment in CLL.

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The biosynthesis of pigments (carotenoids and bacteriochlorophylls) in the photosynthetic bacterium Rhodobacter capsulatus is regulated by the oxygen concentration in the environment. However, the mechanism of this regulation has remained obscure. In this study, transcriptional fusions of the bchCXYZ promoter region to lacZ were used to identify the promoter and regulatory sequences governing transcription of these bacteriochlorophyll biosynthesis genes. The promoter region was identified in vivo by making deletions and site-directed mutations. The 50 bp upstream of the promoter region was shown to be required for the oxygen-dependent transcriptional regulation of bchCXYZ. A previously described palindrome sequence is also likely involved in the regulation. A gel mobility shift assay further defined the interaction of transcription regulators with these DNA sequence elements in vitro and demonstrated that a DNA-protein complex is formed at this promoter region. Since the suggested promoter sequence and the palindrome sequence are found upstream of several other bch and crt operons, these sequences may be responsible for regulating oxygendependent pigment biosynthesis at the level of transcription in R. capsulatus. In addition, these cis-acting DNA elements are not found upstream of puh and puf operons, which encode the structural polypeptides of the reaction center and light-harvesting I complexes. This observation supports the model of different regulatory mechanisms for the pigment biosynthesis enzymes and structural polypeptides required for the production of the photosynthetic apparatus.Rhodobacter capsulatus is a purple nonsulfur bacterium which can grow by multiple metabolic modes (12,19). When the concentration of dissolved oxygen in the environment is low, this bacterium forms an intracytoplasmic photosynthetic membrane that can transduce light energy into chemical energy. The photosynthetic apparatus consists of three major pigment-protein complexes, including a reaction center and two types of light-harvesting complexes, I and II. Each of these complexes is composed of several structural polypeptides that bind bacteriochlorophyll and carotenoid pigments. The structural polypeptides are encoded by the puf, puh, and puc operons, and the pigment biosynthesis enzymes are encoded by multiple bch and crt operons.Oxygen tension is a major factor controlling the coordinate expression of these genes. At least part of the oxygendependent regulation of pigment biosynthesis occurs at the transcriptional level (6,20,27 evidence for the differential regulation of photosynthesis genes in R. capsulatus.Although a great deal has been learned recently about the regulatory mechanism of structural polypeptide biosynthesis, much less is known about regulation of pigment biosynthesis. At least part of the reason for this discrepancy is that the pigment biosynthesis genes are induced to a smaller extent (two-to fivefold) at the transcriptional level than those encoding structural polypeptides (10-to 30-fold) (20) and are the...

Background Splenic marginal zone lymphoma (MZL) is a form of indolent B-cell lymphoma that is not well characterized in dogs. Hypothesis/Objectives The purpose of this study was to describe clinical characteristics and outcome in dogs with splenic MZL confirmed by histopathology, immunophenotyping, and molecular clonality assessment. We hypothesized that affected dogs would have prolonged survival time with splenectomy alone. Animals Thirty-four dogs were included. Twenty-nine dogs were diagnosed after splenectomy, and 5 dogs were diagnosed at necropsy. Methods Pathology records were searched for dogs with histologically confirmed splenic MZL. Clinical and outcome data were retrospectively collected by medical record review, and prognostic factors were evaluated. Histopathology was reviewed by a board-certified pathologist, and tissue sections were subjected to immunophenotyping and molecular clonality assessment by PCR. Results Immunohistochemistry confirmed a B-cell phenotype for all dogs. Molecular clonality assessment was performed in 33 of 34 dogs, of which 24 had clonal rearrangement of immunoglobulin (Ig) loci, 3 had pseudoclonal rearrangement, and 6 had polyclonal rearrangement. The overall median survival time (MST) for the 29 dogs that underwent splenectomy was 383 days. The MST for 14 of 29 asymptomatic dogs that underwent splenectomy for MZL was 1,153 days as compared to 309 days for 15/29 dogs with clinical signs referable to splenic MZL (P = .018). Lymph node involvement, hemoabdomen, anemia, chemotherapy, and concurrent malignancy did not affect survival outcome. Conclusions and Clinical Importance Dogs diagnosed with splenic MZL can have prolonged survival with splenectomy alone, without the use of adjuvant chemotherapy. Asymptomatic dogs may have a better survival outcome.

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Erwinia herbicola, a nonphotosynthetic bacterium, is yellow colored due to the accumulation of unusually polar carotenoids, primarily mono-and diglucosides of zeaxanthin. We have cloned and expressed the gene for the enzyme that catalyzes the glucosylation of znthin. The enzyme has an apparent molecular mass of 45 kDa on an SDS/polyacrylamide gel, which is consistent with its calculated molecular mass. In vitro enzymatic activity was demonstrated using UDP-["4C]glucose and eaxanthin as substrates. The product zeaanthin diglucoside and its intermediate monoglucoside were identified by thin layer chromatography. The optimum pH and temperature ranges of the enzyme are 7.0-7.5 and 32-3TC, respectively. A hydropathy plot indicates no apparent membrane-spanning regions, and biochemical experiments suggest that the enzyme is weakly membrane-associated. The amino acid sequence derived from the zeaxanthin glucosyltransferase gene shows a small region of high similarity with other glucuronosyl-and glucosyltransferases that use either UDP-activated glucuronic acid or a sugar as one of their substrates. Based on these similarities, we propose that this conserved sequence is part of the UDP binding site.

Therapeutic study, Level III (retrospective comparative study). See the Guidelines for Authors for a complete description of levels of evidence.

The cyclisation of lycopene to p-carotene and the hydroxylation of B-carotene to zeaxanthin are common enzymatic steps in the biosynthesis of carotenoids in a wide range of bacteria, fungi, and plants. We have individually expressed in E. coli the two genes coding for these enzymatic steps in Erwinio herbicola. The cyclase and hydroxylase enzymes have apparent molecular weights of 43 kDa and 22 kDa, respectively, as determined by SDS-PAGE. Hydroxylase in vitro activity was obtained only in the cytoplasmic fraction. Cyclase also demonstrated enzyme activity in a crude cell-free lysate, although to a lesser extent.