Combined copy number and mutation analysis identifies oncogenic pathways associated with transformation of follicular lymphoma.

Follicular lymphoma (FL) is typically an indolent disease, but 30-40% of FL cases transform into an aggressive lymphoma (tFL) with a poor prognosis. To identify the genetic changes that drive this transformation, we sequenced the exomes of 12 cases with paired FL and tFL biopsies and identified 45 recurrently mutated genes in the FL-tFL data set and 39 in the tFL cases. We selected 496 genes of potential importance in transformation and sequenced them in 23 additional tFL cases. Integration of the mutation data with copy-number abnormality (CNA) data provided complementary information. We found recurrent mutations of miR-142, which has not been previously been reported to be mutated in FL/tFL. The genes most frequently mutated in tFL included KMT2D (MLL2), CREBBP, EZH2, BCL2 and MEF2B. Many recurrently mutated genes are involved in epigenetic regulation, the Janus-activated kinase-signal transducer and activator of transcription (STAT) or the nuclear factor-κB pathways, immune surveillance and cell cycle regulation or are TFs involved in B-cell development. Of particular interest are mutations and CNAs affecting S1P-activated pathways through S1PR1 or S1PR2, which likely regulate lymphoma cell migration and survival outside of follicles. Our custom gene enrichment panel provides high depth of coverage for the study of clonal evolution or divergence.

High prevalence of adrenal insufficiency in patients with sickle cell disease: results from a community hospital in the U.S.

OBJECTIVE: Despite the low prevalence (0.008%) of adrenal insufficiency (AI) in the general population, this disorder was recently diagnosed in a substantial number of sickle cell disease (SCD) patients at our hospital. The main objective of this study was to assess the prevalence of AI in SCD patients. METHODS: All adult patients admitted to the Department of Medicine at Interfaith Medical Center from October 2010 to November 2011 were eligible for this retrospective study. Medical records of adult SCD patients hospitalized for painful crisis and who had undergone cosyntropin testing were reviewed. Adult non-SCD patients hospitalized for painful crisis and who had undergone cosyntropin testing served as controls. The result of the cosyntropin test was the primary outcome. The prevalence of positive cosyntropin tests was compared between the 2 groups by using Student's t-test, and odds ratios. RESULTS: 62 adult SCD patients were enrolled in the study. 15 underwent cosyntropin testing and 12 (19.4%) of these patients were found to have AI. AI was also diagnosed in 1 of 1,340 non-SCD patients. The odds ratio for AI in SCD to non-SCD patients [(12/62)/(1,340)] was 259. The odds ratio for the prevalence of AI in SCD patients in our study (19.4%) vs. the general population (approximately 0.008%) was 2,375. CONCLUSION: AI occurred in 19.4% of SCD patients included in this study. These patients thus have a 2,375-fold higher risk of developing AI than the general population, and a 259-fold greater risk of developing AI than do hospitalized non-SCD patients.

Gastric marginal zone B cell lymphoma of the duodenum.

Small bowel lymphomas of the extranodal type occur in the young and are characteristically associated with malabsorption syndrome. We present the case of an elderly in whom there was no malabsorption and the duodenal tumor was a gastric type marginal zone B cell lymphoma also known as gastric mucosa-associated lymphoid tissue (MALT) lymphoma. A 73-year-old woman presented to the emergency room with 2 weeks of general weakness, recurrent vomiting containing food particles and abdominal distension. She had been diagnosed with diabetic gastroparesis 4 years prior. CT of the abdomen and pelvis was suggestive of gastric outlet obstruction but no evidence of pancreatic or duodenal mass. Endoscopy and biopsy of the tumor obstructing the distal first part of the duodenum confirmed a gastric marginal MALT lymphoma. The patient's symptoms improved with radiotherapy. Gastric MALT lymphoma, an extranodal lymphoma primarily described in the stomach, can also present in the small bowel and is not associated with malabsorption.

Epstein-Barr virus-associated lymphoproliferative disease in non-immunocompromised hosts: a status report and summary of an international meeting, 8-9 September 2008.

BACKGROUND: Recently novel Epstein-Barr virus (EBV) lymphoproliferative diseases (LPDs) have been identified in non-immunocompromised hosts, both in Asia and Western countries. These include aggressive T-cell and NK-cell LPDs often subsumed under the heading of chronic active Epstein-Barr virus (CAEBV) infection and EBV-driven B-cell LPDs mainly affecting the elderly. DESIGN: To better define the pathogenesis, classification, and treatment of these disorders, participants from Asia, The Americas, Europe, and Australia presented clinical and experimental data at an international meeting. RESULTS: The term systemic EBV-positive T-cell LPD, as adopted by the WHO classification, is preferred as a pathological classification over CAEBV (the favored clinical term) for those cases that are clonal. The disease has an aggressive clinical course, but may arise in the background of CAEBV. Hydroa vacciniforme (HV) and HV-like lymphoma represent a spectrum of clonal EBV-positive T-cell LPDs, which have a more protracted clinical course; spontaneous regression may occur in adult life. Severe mosquito bite allergy is a related syndrome usually of NK cell origin. Immune senescence in the elderly is associated with both reactive and neoplastic EBV-driven LPDs, including EBV-positive diffuse large B-cell lymphomas. CONCLUSION: The participants proposed an international consortium to facilitate further clinical and biological studies of novel EBV-driven LPDs.

Stromal gene signatures in large-B-cell lymphomas.

BACKGROUND: The addition of rituximab to combination chemotherapy with cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP), or R-CHOP, has significantly improved the survival of patients with diffuse large-B-cell lymphoma. Whether gene-expression signatures correlate with survival after treatment of diffuse large-B-cell lymphoma is unclear. METHODS: We profiled gene expression in pretreatment biopsy specimens from 181 patients with diffuse large-B-cell lymphoma who received CHOP and 233 patients with this disease who received R-CHOP. A multivariate gene-expression-based survival-predictor model derived from a training group was tested in a validation group. RESULTS: A multivariate model created from three gene-expression signatures--termed "germinal-center B-cell," "stromal-1," and "stromal-2"--predicted survival both in patients who received CHOP and patients who received R-CHOP. The prognostically favorable stromal-1 signature reflected extracellular-matrix deposition and histiocytic infiltration. By contrast, the prognostically unfavorable stromal-2 signature reflected tumor blood-vessel density. CONCLUSIONS: Survival after treatment of diffuse large-B-cell lymphoma is influenced by differences in immune cells, fibrosis, and angiogenesis in the tumor microenvironment.

Light-chain-restricted germinal centres in reactive lymphadenitis: report of eight cases.

AIMS: Light-chain-restricted germinal centres are generally associated with the existence of a neoplastic lymphoproliferative disorder. The aim was to present a series of cases with persistent lymph node enlargement that featured some germinal centres showing light chain immunoglobulin restriction. METHODS AND RESULTS: A series of six reactive lymphadenitis and two Castleman's disease cases was analysed by immunohistochemistry, IgH-polymerase chain reaction (PCR) and microdissected PCR. In all cases some germinal centres contained a population of plasma cells and plasmacytoid germinal centre cells showing light chain immunoglobulin restriction. In three cases the monotypic cells also showed distinct Bcl-2 expression. Two of the cases showed a predominant IgH rearrangement on a florid polyclonal background and one had an IgH monoclonal rearrangement, as revealed by PCR. Microdissected germinal centre PCR revealed a dominant repeated band in one of three cases and in another case a non-repeated clonal peak was observed. One of the patients developed a follicular lymphoma, which became evident from a subsequent biopsy. CONCLUSIONS: These findings may be a manifestation of an underlying disorder in the regulation of the immune response, or an exaggeration of the germinal centre oligoclonal nature. This should be taken into account in the differential diagnosis of follicular hyperplasia.

Distinctive patterns of BCL6 molecular alterations and their functional consequences in different subgroups of diffuse large B-cell lymphoma.

Gene expression profiling of diffuse large B-cell lymphoma (DLBCL) has revealed biologically and prognostically distinct subgroups: germinal center B-cell-like (GCB), activated B-cell-like (ABC) and primary mediastinal (PM) DLBCL. The BCL6 gene is often translocated and/or mutated in DLBCL. Therefore, we examined the BCL6 molecular alterations in these DLBCL subgroups, and their impact on BCL6 expression and BCL6 target gene repression. BCL6 translocations at the major breakpoint region (MBR) were detected in 25 (18.8%) of 133 DLBCL cases, with a higher frequency in the PM (33%) and ABC (24%) subgroups than in the GCB (10%) subgroup. Translocations at the alternative breakpoint region (ABR) were detected in five (6.4%) of 78 DLBCL cases, with three cases in ABC and one case each in the GCB and the unclassifiable subgroups. The translocated cases involved IgH and non-IgH partners in about equal frequency and were not associated with different levels of BCL6 mRNA and protein expression. BCL6 mutations were detected in 61% of DLBCL cases, with a significantly higher frequency in the GCB and PM subgroups (>70%) than in the ABC subgroup (44%). Exon-1 mutations were mostly observed in the GCB subgroup. The repression of known BCL6 target genes correlated with the level of BCL6 mRNA and protein expression in GCB and ABC subgroups but not with BCL6 translocation and intronic mutations. No clear inverse correlation between BCL6 expression and p53 expression was observed. Patients with higher BCL6 mRNA or protein expression had a significantly better overall survival. The biological role of BCL6 in translocated cases where repression of known target genes is not demonstrated is intriguing and warrants further investigation.

Depolymerisation and rearrangement of actin filaments during exocytosis in rat peritoneal mast cells: involvement of ryanodine-sensitive calcium stores.

Cytoskeletal F-actin associated with synaptic vesicles and granules plays an important role during Ca(2+)-mediated exocytosis. In the present work, we have used amperometry and confocal fluorescence to study the role of internal Ca(2+) in the rearrangement of F-actin (visualised with phalloidin-Alexa 546) during exocytosis in rat mast cells. The F-actin-depolymerising drug, latrunculin A, and the ryanodine receptor agonists ryanodine and caffeine that, per se did not induce exocytosis, enhanced the exocytotic responses elicited by compound 48/80 (C48/80). They also induced cortical actin depolymerisation in the presence or absence of external Ca(2+). Degranulation induced by C48/80 was accompanied by the formation of a cytoplasmic F-actin network. Depletion of internal Ca(2+) with cyclopiazonic acid inhibited latrunculin potentiation of C48/80-stimulated exocytosis and completely blocked the formation of the cytoplasmic F-actin network. This indicates that the mobilisation of Ca(2+) from ryanodine-sensitive intracellular stores plays an important role in the depolymerisation of the cortical F-actin barrier and possibly in the formation of the internal F-actin network during exocytotic activation of peritoneal mast cells.

A practical approach to pediatric patients referred with an abnormal coagulation profile.

CONTEXT: Workup for prolonged prothrombin time (PT) and activated partial thromboplastin time (PTT) is a frequent referral to a Hematology and Coagulation Laboratory. Although the workup should be performed in a timely and cost-effective manner, the complete laboratory assessment of the coagulation state has not been standardized. OBJECTIVE: To determine which clinical and laboratory data are most predictive of a coagulopathy and to formulate the most efficient strategy to reach a diagnosis in patients referred for abnormal coagulation profiles. DESIGN: Retrospective case review. Medical records of 251 patients referred for prolonged PT and/or PTT to our Hematology Service between June 1995 and December 2002 were reviewed. RESULTS: The study included 135 males and 116 females with a mean age of 7.0 years. A personal history of bleeding was reported in 137 patients, and a family history of bleeding was reported in 116 patients. Fifty-one patients (20%) had a coagulopathy (ie, a bleeding risk). Factors predictive of a bleeding risk were a positive family history of bleeding (P < .001) and a positive personal history of bleeding (P = .001). Of 170 patients with findings of normal PT and PTT values on repeat testing, 14 were subsequently diagnosed with a coagulopathy. Two of these patients reported no positive personal or family history of bleeding. CONCLUSIONS: Coagulopathy was identified in 20% of the children referred for abnormal PT and/or PTT. In the absence of a personal or family history of bleeding, a normal PT and/or PTT on repeat testing has a negative predictive value of more than 95%.

Tumours of histiocytes and accessory dendritic cells: an immunohistochemical approach to classification from the International Lymphoma Study Group based on 61 cases.

Neoplasms of histiocytes and dendritic cells are rare, and their phenotypic and biological definition is incomplete. Seeking to identify antigens detectable in paraffin-embedded sections that might allow a more complete, rational immunophenotypic classification of histiocytic/dendritic cell neoplasms, the International Lymphoma Study Group (ILSG) stained 61 tumours of suspected histiocytic/dendritic cell type with a panel of 15 antibodies including those reactive with histiocytes (CD68, lysozyme (LYS)), Langerhans cells (CD1a), follicular dendritic cells (FDC: CD21, CD35) and S100 protein. This analysis revealed that 57 cases (93%) fit into four major immunophenotypic groups (one histiocytic and three dendritic cell types) utilizing six markers: CD68, LYS, CD1a, S100, CD21, and CD35. The four (7%) unclassified cases were further classifiable into the above four groups using additional morphological and ultrastructural features. The four groups then included: (i) histiocytic sarcoma (n=18) with the following phenotype: CD68 (100%), LYS (94%), CD1a (0%), S100 (33%), CD21/35 (0%). The median age was 46 years. Presentation was predominantly extranodal (72%) with high mortality (58% dead of disease (DOD)). Three had systemic involvement consistent with 'malignant histiocytosis'; (ii) Langerhans cell tumour (LCT) (n=26) which expressed: CD68 (96%), LYS (42%), CD1a (100%), S100 (100%), CD21/35 (0%). There were two morphological variants: cytologically typical (n=17) designated LCT; and cytologically malignant (n=9) designated Langerhans cell sarcoma (LCS). The LCS were often not easily recognized morphologically as LC-derived, but were diagnosed based on CD1a staining. LCT and LCS differed in median age (33 versus 41 years), male:female ratio (3.7:1 versus 1:2), and death rate (31% versus 50% DOD). Four LCT patients had systemic involvement typical of Letterer-Siwe disease; (iii) follicular dendritic cell tumour/sarcoma (FDCT) (n=13) which expressed: CD68 (54%), LYS (8%), CD1a (0%), S100 (16%), FDC markers CD21/35 (100%), EMA (40%). These patients were adults (median age 65 years) with predominantly localized nodal disease (75%) and low mortality (9% DOD); (iv) interdigitating dendritic cell tumour/sarcoma (IDCT) (n=4) which expressed: CD68 (50%), LYS (25%), CD1a (0%), S100 (100%), CD21/35 (0%). The patients were adults (median 71 years) with localized nodal disease (75%) without mortality (0% DOD). In conclusion, definitive immunophenotypic classification of histiocytic and accessory cell neoplasms into four categories was possible in 93% of the cases using six antigens detected in paraffin-embedded sections. Exceptional cases (7%) were resolvable when added morphological and ultrastructural features were considered. We propose a classification combining immunophenotype and morphology with five categories, including Langerhans cell sarcoma. This simplified scheme is practical for everyday diagnostic use and should provide a framework for additional investigation of these unusual neoplasms.

Macrophage-derived chemokine expression in classical Hodgkin's lymphoma: application of tissue microarrays.

Hodgkin's disease (HD) is a lymphoid malignancy characterized by the presence of Reed-Sternberg (RS) and Hodgkin's cells in a background of mixed inflammatory cells and stromal reaction. Studies have documented that HD is a neoplasm associated with abnormal cytokine and chemokine production. To define the expression of macrophage-derived chemokine (MDC) in HD, 57 cases (18 lymphocyte predominant, 11 mixed cellularity, 28 nodular sclerosis) were stained for MDC by immunohistochemistry and compared with reactive lymph nodes as controls. MDC was expressed by RS cells in classical HD (CHD) and showed a distinct cytoplasmic and Golgi localization. Accumulating evidence suggests that lymphocyte-predominant HD (LPHD) represents an entity distinct from CHD, with different biological properties and clinical course. On the basis of the high level of MDC staining alone, CHD could be distinguished from LPHD (P <.001), which showed only faint staining of scattered histiocytes similar to control tissues. CHD cases with high MDC mRNA levels showed high levels of MDC protein expression by immunohistochemistry (P <.001) and significant eosinophil infiltration, suggesting that MDC may represent another molecule that plays a critical role in eosinophil recruitment. We also analyzed 102 cases of non-Hodgkin's lymphoma and normal spleen, lymph node, and thymic tissue. High levels of MDC expression were specific to CHD cases because only low levels of MDC were observed in a minor subset of LPHD, NHL or normal lymphoid tissues.

Cutaneous manifestations of lymphoma: a clinical guide based on the WHO classification. World Health Organization.

Cutaneous lymphomas are a heterogeneous group of lymphomas that show variations in histology, immunophenotype, and prognosis. At the time of presentation, cutaneous lymphomas may be primary or may involve the skin as a secondary site of involvement. Primary cutaneous lymphomas, in many instances, are distinct from morphologically similar lymphomas arising in lymph nodes. Their natural history is often more indolent than nodal lymphomas, and for that reason, they often require different therapeutic approaches. A classification scheme should recognize those lymphomas that are unique to the skin, as well as those arising in other sites. The mode of presentation of a lymphoma is often an indication of underlying biological distinctions. However, organ-specific classification systems undermine communication among medical specialists. The World Health Organization classification of hematopoietic and lymphoid malignancies offers a comprehensive approach and proposes that lymphomas should be viewed as a list of individual diseases and that each disease can be defined by a constellation of morphological, biological, and clinical features. The current review will focus on the spectrum of primary and secondary cutaneous lymphomas, emphasizing those features of importance to the clinical oncologist.

Cutaneous lymphomatoid granulomatosis: correlation of clinical and biologic features.

Lymphomatoid granulomatosis (LYG) is a rare angiocentric and angiodestructive Epstein-Barr virus-associated B-cell lymphoproliferative disorder (EBV-BLPD), varying widely from an indolent process to an aggressive large cell lymphoma. The skin is the extrapulmonary organ most commonly involved in LYG. We studied 32 skin lesions from 20 patients with known pulmonary LYG, using immunohistochemistry, in situ hybridization for EBV, and polymerase chain reaction for the presence of antigen receptor gene rearrangements (IgH and TCR) to better define both the clinicopathologic spectrum and pathogenesis of the cutaneous lesions. We describe two distinct patterns of cutaneous involvement. Multiple erythematous dermal papules and/or subcutaneous nodules, with or without ulceration, were present in 17 patients (85%). These lesions demonstrate a marked angiocentric lymphohistiocytic infiltrate, composed predominantly of CD4-positive T-cells, with a high propensity for involving the subcutaneous tissues, and exhibiting angiodestruction, necrosis, and cytologic atypia. EBV-positive B-cells were detected in the nodules from five patients; clonal immunoglobulin heavy chain gene (IgH) rearrangements were detected by polymerase chain reaction in two patients. Multiple indurated, erythematous to white plaques were present in three patients (15%). The plaque lesions were negative for EBV and clonal IgH gene rearrangements in all cases studied. The clinical course of overall disease was variable, ranging from spontaneous regression without treatment (1 of 13; 7%), resolution with chemo/immunomodulatory therapy (8 of 13; 62%), and progression (4 of 13; 31%). The clinical and histopathologic features of cutaneous LYG are extremely diverse. However, the majority (85%) of the cutaneous lesions mirrors to some extent LYG in the lung, although EBV+ cells are less frequently identified. This subset of cases shows the histopathologic triad of angiodestruction with associated necrosis, panniculitis, and in some cases atypical lymphoid cells. The commonality of the histologic features in this group suggests a common pathophysiologic basis, possibly mediated by cytokines and chemokines induced by EBV. A small percentage of the lesions (15%) presented as indurated and atrophic plaques, and EBV was not identified in the small number of cases studied. The relationship of the plaque-like lesions to LYG remains uncertain. Whereas some cases of LYG regress spontaneously, most require therapy.

Regression and clonally distinct recurrence of human immunodeficiency virus related Burkitt-like lymphoma during antiretroviral therapy.

An increased incidence of intermediate to high-grade Non Hodgkin's Lymphoma is found in individuals with AIDS. Although immune function in AIDS patients can be improved through the use of antiretroviral therapy, the contribution of these drugs to lymphoma regression is not known. Here we describe the complete regression and subsequent recurrence of high grade, Burkitt-like lymphoma during antiretroviral therapy in a patient with AIDS. Antiretroviral therapy resulted in diminished viral load and modest improvement in CD4+ T cell counts. Lymphoma regressed initially, but relapsed 3 months later. Tissue taken from the initial and recurrent tumor demonstrated different clonal rearrangements. The recurrent lymphoma did not respond to continued antiretroviral therapy. In Conclusion, antiretroviral therapy may contribute to lymphoma regression in AIDS lymphoma. Clinically recurrent disease may be clonally distinct.

Burkitt's/Burkitt's-like lymphoma presenting as bacterial sinusitis in two HIV-infected children.

Two children (ages 12 and 13 years) with transfusion-acquired human immunodeficiency virus (HIV) infection presented with facial pain and rhinorrhea. Radiographic imaging showed extensive paranasal sinus disease, presumed to be bacterial sinusitis, and the patients were treated with broad-spectrum oral antibiotics. Both patients were unresponsive to oral agents and were switched to intravenous antibiotics. Despite aggressive antimicrobial therapy, one patient (case 1) developed increased periorbital swelling and proptosis, and the other patient (case 2) developed symptoms of nasopharyngeal obstruction. Repeat imaging showed progression of the infiltrative process extending from the paranasal sinuses into the orbit (case 1), and nasopharynx (case 2). Surgical exploration and tissue biopsies were performed on both patients and the histopathology was consistent with Burkitt's/Burkitt's-like lymphoma. Combination systemic and intrathecal chemotherapy resulted in a complete remission in both patients. These reports illustrate the fact that Burkitt's/Burkitt's-like lymphoma in the paranasal sinuses may initially masquerade as an acute bacterial sinusitis. The ability of the tumor to extend rapidly from the sinuses into the orbit and nasopharynx reinforces the importance of early diagnosis and treatment. Burkitt's/Burkitt's-like lymphoma in the paranasal sinuses has not been previously described in HIV-infected children.

Chemokine gene expression and clonal analysis of B cells in tissues involved by lymphoid interstitial pneumonitis from HIV-infected pediatric patients.

Lymphoid interstitial pneumonitis (LIP), a frequent pulmonary complication in HIV-infected pediatric patients, is characterized histologically by marked infiltration of lymphoid cells. We sought to evaluate the nature and pathogenesis of the lymphoid infiltrates and to examine the relationship of LIP to pulmonary MALT lymphoma that has been described in pediatric HIV positive patients. To examine the potential contribution of chemokines and cytokines to the inflammatory cell recruitment in tissues involved by lymphoid interstitial pneumonitis from HIV-infected pediatric patients, RNA was extracted from paraffin-embedded tissues from five lung biopsies in four pediatric HIV-positive patients and from five control, normal lung biopsies in five HIV-negative patients and was analyzed by semiquantitative RT-PCR for the expression of cytokines (TNF-alpha, GM-CSF, IFN-gamma, IL-4, IL-6, IL-10, and IL-18) and chemokines (IP-10, Mig, regulated upon activation, normal T expressed and secreted [RANTES], and MIP1-alpha and beta) after normalization for G3PDH. Expression of IL-18 was increased, as well as expression of IFN-gamma-inducible chemokines IP-10 and Mig in LIP tissues compared with controls. RANTES and MIP1-alpha and -beta were also increased in pediatric LIP lesions compared with controls. In contrast, expression of TNF-alpha, GM-CSF, IL-10, and IL-6 was variable in LIP tissues and controls. In addition, clonality of the B-cell population was evaluated by VDJ-PCR. A polyclonal B-cell population was shown in all five biopsies from five patients with LIP; and in one patient with concurrent LIP and MALT lymphoma, a band of increased intensity was observed in the LIP biopsy that was identical in size to the monoclonal band in the concurrent MALT lymphoma biopsy. These results provide evidence of high-level expression of certain chemokines in lymphoid interstitial pneumonitis tissues and suggest that chemokines and cytokines may play an important role in the recruitment of inflammatory cell infiltrates into these tissues. In addition, LIP may represent an early stage of MALT lymphoma or an immunologic response to a chronic antigenic stimulus that may provide a milieu or microenvironment for the evolution of a monoclonal B-cell population.

Mechanistic basis for suicide inactivation of porphobilinogen synthase by 4,7-dioxosebacic acid, an inhibitor that shows dramatic species selectivity.

4,7-Dioxosebacic acid (4,7-DOSA) is an active site-directed irreversible inhibitor of porphobilinogen synthase (PBGS). PBGS catalyzes the first common step in the biosynthesis of the tetrapyrrole cofactors such as heme, vitamin B(12), and chlorophyll. 4,7-DOSA was designed as an analogue of a proposed reaction intermediate in the physiological PBGS-catalyzed condensation of two molecules of 5-aminolevulinic acid. As shown here, 4,7-DOSA exhibits time-dependent and dramatic species-specific inhibition of PBGS enzymes. IC(50) values vary from 1 microM to 2.4 mM for human, Escherichia coli, Bradyrhizobium japonicum, Pseudomonas aeruginosa, and pea enzymes. Those PBGS utilizing a catalytic Zn(2+) are more sensitive to 4,7-DOSA than those that do not. Weak inhibition of a human mutant PBGS establishes that the inactivation by 4,7-DOSA requires formation of a Schiff base to a lysine that normally forms a Schiff base intermediate to one substrate molecule. A 1.9 A resolution crystal structure of E. coli PBGS complexed with 4,7-DOSA (PDB code ) shows one dimer per asymmetric unit and reveals that the inhibitor forms two Schiff base linkages with each monomer, one to the normal Schiff base-forming Lys-246 and the other to a universally conserved "perturbing" Lys-194 (E. coli numbering). This is the first structure to show inhibitor binding at the second of two substrate-binding sites.