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Purpose: Resistance to temozolomide chemotherapy is partly mediated by O(6)-methylguanine-DNA methlytransferase (MGMT). Protracted treatment with temozolomide potentially overcomes MGMT resistance and improves outcome. We conducted a phase II study of protracted daily temozolomide in adults with low-grade gliomas., Experimental Design: Patients with newly diagnosed oligodendroglioma or oligoastrocytoma with a MIB-1 index of >5% or recurrent low-grade gliomas received temozolomide (75 mg/m(2)/day in 11-week cycles of 7 weeks on/4 weeks off). Treatment continued for a total of six cycles or until tumor progression or unacceptable toxicity. Primary end point was best overall response rate; secondary end points were progression-free survival, overall survival, and toxicity. We correlated response with MGMT promoter methylation and chromosome 1p/19q deletion status., Results: Forty-four patients were treated (14 female, 30 male) with a median follow-up of 39.4 months. Median age was 43 years (range, 20-68 years) and median Karnofsky performance status was 90 (range, 70-100). The regimen was well tolerated. No patients had a complete response (0%), 9 had partial response (20%), 33 had stable disease (75%), and 2 had progressive disease (5%). A total of 21 patients eventually progressed with an overall median progression-free survival of 38 months. Patients with methylated MGMT promoter had a longer overall survival (P = 0.008). Deletion of either 1p or 19q chromosomes also predicted longer overall survival (hazard ratio, 0.17; 95% confidence interval, 0.03-0.93; log-rank P = 0.02)., Conclusions: A protracted course of daily temozolomide is a well-tolerated regimen and seems to produce effective tumor control. This compares favorably with historical data on the standard 5-day temozolomide regimen.

Regardless of the morphological divergence among larval forms of marine bryozoans, the larval nervous system and its major effector organs (musculature and ciliary fields) are largely molded on the basis of functional demands of feeding, ciliary propulsion, phototactic behaviors, and substrate exploration. Previously published ultrastructural information and immunohistochemical reconstructions presented here indicate that neuronal pathways are largely ipsilateral, with more complex synaptic connections localized within the nerve nodule. Multiciliated sensory-motor neurons diversify structurally and functionally on the basis of their position along the axis of swimming largely due to the functional demands of photoklinotaxis and substrate exploration. Vesiculariform, buguliform, and ascophoran coronate larvae all have patches of sensory neurons bordering the pyriform organ's ciliated groove (juxtapapillary cells and border cells) that are active during substrate selection. Despite their simplified form, cyclostome larvae maintain swimming and probing behaviors with sensory-motor systems functionally similar to those of some parenchymella and planula larval types. Considering the evolutionary relationships among the morphological grades of marine bryozoans, particular lineages within the gymnolaemates have independently evolved larval traits that convey a greater range of sensory abilities and increased propulsive capacity. The larval nervous system of bryozoans may be evolutionarily derived from the pretrochal region of a trochophore-like larval form.

Despite the embryological and anatomical disparities present among lophotrochozoan phyla, there are morphological similarities in the cellular arrangements of ciliated cells used for propulsion among the nonfeeding larval forms of kamptozoans, nemerteans, annelids, mollusks, and bryozoans. Evaluating whether these similarities are the result of convergent selective pressures or a shared (deep) evolutionary history is hindered by the paucity of detailed cellular information from multiple systematic groups from lesser-known, and perhaps, basal evolutionary phyla such as the Bryozoa. Here, I compare the ciliary fields and musculature among the major morphological grades of marine bryozoan larvae using light microscopy, SEM, and confocal imaging techniques. Sampling effort focused on six species from systematic groups with few published accounts, but an additional four well-known species were also reevaluated. Review of the main larval types among species of bryozoans and these new data show that, within select systematic groups of marine bryozoans, there is some conservation of the cellular arrangement of ciliary fields and larval musculature. However, there is much more morphological diversity in these structures than previously documented, especially among nonfeeding ctenostome larval types. This structural and functional diversification reflects species differences in the orientation of the apical disc during swimming and crawling behaviors, modification of the presumptive juvenile tissues, elongation of larval forms in the aboral-oral axis, maximizing the surface area of cell types with propulsive cilia, and the simplification of ciliary fields and musculature within particular lineages due to evolutionary loss. Considering the embryological origins and functional plasticity of ciliated cells within bryozoan larvae, it is probable that the morphological similarities shared between the coronal cells of bryozoan larvae and the prototrochal cells of trochozoans are the result of convergent functional solutions to swimming in the plankton. However, this does not rule out cell specification pathways shared by more closely related spiralian phyla. Overall, among the morphological grades of larval bryozoans, the structural variation and arrangement of the main cell groups responsible for ciliary propulsion have been evolutionarily decoupled from the more divergent modifications of larval musculature. The structure of larval ciliary fields reflects the functional demands of swimming and substrate exploration behaviors before metamorphosis, but this is in contrast to the morphology of larval musculature and presumptive juvenile tissues that are linked to macroevolutionary differences in morphogenetic movements during metamorphosis.

Tyrosine hydroxylase deficiency, a cause of the autosomal recessive form of L-DOPA responsive dystonia, has been associated with a broad spectrum of movement disorders and clinical courses. We describe a new patient presenting with an early onset spastic paraplegia who later developed a progressive generalized dystonic-dyskinetic syndrome. He markedly improved with a very low dosage of L-DOPA/carbidopa, while higher dosages were not tolerated. Two novel mutations (p.G414R/p.L510Q) were detected in the TH gene.

The core embryonic stem cell transcription factors (TFs) OCT3/4 (OCT4), NANOG, and SOX2 have shared as well as nonoverlapping roles in stem cell growth and differentiation. These same TFs are also expressed in various types of human germ cell tumors (GCTs), implicating them in regulation of tumor growth and differentiation. Although NANOG and OCT3/4 are sensitive and specific markers for seminoma and embryonal carcinoma, neither factor aids in the clinically important distinction of seminomatous from nonseminomatous tumors. In contrast, expression profiling data suggest that SOX2 may help with this distinction. To determine if a panel of embryonic stem cell TFs (NANOG, OCT3/4, and SOX2) can facilitate the identification and distinction of seminomatous from nonseminomatous GCTs, we evaluated their expression by immunohistochemistry in primary testicular (n=41) and metastatic retroperitoneal (n=43) GCTs. Our results confirm NANOG and OCT3/4 as sensitive and specific markers for primary seminoma and embryonal carcinoma and demonstrate the novel finding that NANOG is a marker for metastatic GCTs. In addition, SOX2 is expressed in embryonal carcinoma but not pure seminoma and is therefore a useful diagnostic marker for distinguishing seminomatous and nonseminomatous GCTs. In summary, we find that the embryonic stem cell TF signature of seminoma is NANOG+, OCT3/4+, and SOX2-, whereas embryonal carcinoma is NANOG+, OCT3/4+, and SOX2+, and expect these immunohistochemical profiles will facilitate the diagnosis of both primary and metastatic GCTs.

The homeodomain transcription factor, NANOG, along with OCT3/4 (POU5F1) and SOX2, is part of the core set of transcription factors that maintain embryonic stem cell self-renewal and pluripotency. Expression of NANOG has been detected in fetal germ cells and in gonadal germ cell tumors. To assess the diagnostic utility of NANOG in central nervous system (CNS) germ cell tumors, we analyzed its expression by immunohistochemistry in a series of 12 CNS germinomas and compared its expression with other stem cell markers. Strong nuclear expression of NANOG was demonstrated in >90% of the tumor cells in all cases. In contrast, expression of OCT3/4 and placental alkaline phosphatase was inconsistent and SOX2 was expressed in only rare cells. NANOG was not detected in tumor types frequently considered in the differential diagnosis of CNS germinoma: pineoblastoma, primitive neuroectodermal tumors, medulloblastoma, lymphoma, pituitary adenoma, atypical teratoid/rhabdoid tumor, Langerhans cell histiocytosis, and gliomas. These findings demonstrate that NANOG is a sensitive and specific marker of CNS germinoma. Compared with other currently used markers, NANOG may have superior diagnostic characteristics and can facilitate identification of germinomas in minute surgical biopsies commonly obtained from these tumors. These findings also suggest a potential biologic role for NANOG in maintenance of CNS germinoma.

Metamorphosis (Gr. meta- "change" + morphe "form") as a biological process is generally attributed to a subset of animals: most famously insects and amphibians, but some fish and many marine invertebrates as well. We held a symposium at the 2006 Society for Integrative and Comparative Biology (SICB) annual meeting in Orlando, FL (USA) to discuss metamorphosis in a comparative context. Specifically, we considered the possibility that the term "metamorphosis" could be rightly applied to non-animals as well, including fungi, flowering plants, and some marine algae. Clearly, the answer depends upon how metamorphosis is defined. As we participants differed (sometimes quite substantially) in how we defined the term, we decided to present each of our conceptions of metamorphosis in 1 place, rather than attempting to agree on a single consensus definition. Herein we have gathered together our various definitions of metamorphosis, and offer an analysis that highlights some of the main similarities and differences among them. We present this article not only as an introduction to this symposium volume, but also as a reference tool that can be used by others interested in metamorphosis. Ultimately, we hope that this article-and the volume as a whole-will represent a springboard for further investigations into the surprisingly deep mechanistic similarities among independently evolved life cycle transitions across kingdoms.

Neuromas typically arise in the peripheral nervous system in response to traumatic injury at the site of partial or complete nerve transection as new axons from the proximal nerve stump sprout to reinnervate the distal segment. In rare cases neuromas have also been described as intramedullary spinal cord lesions. These lesions have been identified as incidental autopsy findings in association with prior trauma and cervical spondylosis, multiple sclerosis, spinal tumors, and syringomyelia. The authors report the case of a 50-year-old man who had been involved in a motor vehicle accident, during which his car was struck from behind as it was stationary at an intersection, more than 5 years before presentation. A workup for syncopal and presyncopal episodes involved magnetic resonance imaging that revealed a 1.1-cm lesion at the cervicomedullary junction (CMJ). The imaging features of the lesion raised the question of an ependymoma or subependymoma. The lesion was excised, and examination of the tissue demonstrated a neuroma with haphazardly arranged interlacing bundles of axons ensheathed by Schwann cells with interfascicular regions of reactive glial cells and Rosenthal fibers, consistent with those present after traumatic injury. This case may represent the first true traumatic intramedullary neuroma of the CMJ diagnosed in a living patient and treated surgically.

Background: Guanidinoacetate (GAA) and creatine (Cr) are reliable biochemical markers of primary creatine disorders. The aim of this study was to develop and validate a method for the determination of GAA and Cr in dried blood spot through the use of stable isotope dilution and flow injection analysis (FIA)-ESI-MS/MS., Methods: Dried blood spots were extracted using methanol-water solution containing D3-Cr. After evaporation and formation of butyl esters, samples were analyzed using multiple reaction monitoring mode (m/z 174.2-->101.1 for GAA, 188.3-->90.1 for Cr and 191.3-->93.1 for D3-Cr)., Results: The analysis was very fast (1 min). The detection limits were 0.34 micromol/l of blood and 0.30 micromol/l of blood for Cr and GAA, respectively, and the response was linear over the range 0.25-12.5 micromol/l of blood for GAA and 3.57-624.7 micromol/l of blood for Cr. Recovery range was 93-101% for Cr and 94-105% for GAA and between-run CVs were 5.3% for GAA and 4.5% for Cr. Ion suppression effect was also studied. The method was applied to spots obtained from two patients affected by GAMT deficiency, four patients affected by AGAT deficiency (including a newborn) as well as 282 healthy subjects., Conclusions: The detection of GAA in dried blood spot by FIA-ESI-MS/MS is a highly reliable and high throughput method for the diagnosis of GAMT and AGAT deficiencies and a possible tool for newborn screening of both these tractable disorders.

Pheochromocytomas are neural crest-derived tumors that arise from inherited or sporadic mutations in at least six independent genes. The proteins encoded by these multiple genes regulate distinct functions. We show here a functional link between tumors with VHL mutations and those with disruption of the genes encoding for succinate dehydrogenase (SDH) subunits B (SDHB) and D (SDHD). A transcription profile of reduced oxidoreductase is detected in all three of these tumor types, together with an angiogenesis/hypoxia profile typical of VHL dysfunction. The oxidoreductase defect, not previously detected in VHL-null tumors, is explained by suppression of the SDHB protein, a component of mitochondrial complex II. The decrease in SDHB is also noted in tumors with SDHD mutations. Gain-of-function and loss-of-function analyses show that the link between hypoxia signals (via VHL) and mitochondrial signals (via SDH) is mediated by HIF1alpha. These findings explain the shared features of pheochromocytomas with VHL and SDH mutations and suggest an additional mechanism for increased HIF1alpha activity in tumors., Competing Interests: Competing interests. The authors have declared that no competing interests exist.

Phoronis pallida (Phoronida) occurs as a commensal within the burrow of Upogebia pugettensis (Decapoda: Thalassinidea). Upogebia-conditioned seawater (UCSW) induced an exploratory swimming behavior in competent larvae of P. pallida in a dosage-dependent manner. This behavior included a significant increase in swimming speed that was directed downward, along with the repeated probing of the bottom with the sensory portion of the oral hood. The waterborne cue from the shrimp was present in the gut effluent, and the swimming behavior was not the result of the elevated ammonia concentration. Molecular weight separation of the UCSW estimated that the cue was between 10 and 50 kDa. Enzymatic treatments showed that the cue's activity could be eliminated by arginase and significantly reduced by lipase. Competent larvae were also induced to metamorphose when exposed to 20 mM CsCl for 30 min. Larvae did not respond to CsCl when cultured about 4 weeks past the onset of competence. Compared with actinotroch larvae of other phoronid species, P. pallida larvae exhibit greater behavioral specificity and neuronal differences within the hood sense organ. These anatomical and behavioral differences may have been maintained through a coevolutionary process among P. pallida and species of thalassinid shrimps that share Upogebia life-history characteristics.

Recent studies suggest that NOTCH signaling can promote epithelial-mesenchymal transitions and augment signaling through AKT, an important growth and survival pathway in epithelial cells and prostate cancer in particular. Here we show that JAGGED1, a NOTCH receptor ligand, is significantly more highly expressed in metastatic prostate cancer as compared with localized prostate cancer or benign prostatic tissues, based on immunohistochemical analysis of JAGGED1 expression in human tumor samples from 154 men. Furthermore, high JAGGED1 expression in a subset of clinically localized tumors was significantly associated with recurrence, independent of other clinical parameters. These findings support a model in which dysregulation of JAGGED1 protein levels plays a role in prostate cancer progression and metastasis and suggest that JAGGED1 may be a useful marker in distinguishing indolent and aggressive prostate cancers.

Morphological variation among larval body plans must be placed into a phylogenetic and ecological context to assess whether similar morphologies are the result of phylogenetic constraints or convergent selective pressures. Investigations are needed of the diverse larval forms within the Lophotrochozoa, especially the larvae of phoronids and brachiopods. The actinotroch larva of Phoronis pallida (Phoronida) was reared in the laboratory to metamorphic competence. Larval development and growth were followed with video microscopy, SEM, and confocal microscopy. Early developmental features were similar to other phoronid species. Gastrulation was accomplished by embolic invagination of the vegetal hemisphere. Mesenchymal cells were found in the remaining blastocoelic space after invagination began. Mesenchymal cells formed the body wall musculature during the differentiation of larval features. Body wall musculature served as the framework from which all other larval muscles proliferated. Larval growth correlated best with developmental stage rather than age. Consistent with other phoronid species, differentiation of juvenile tissues occurred most rapidly at the latest stages of larval development. The minimum precompetency period of P. pallida was estimated to be approximately 4-6 weeks. Previously published studies have documented that the planktonic embryos of P. pallida develop faster than the brooded embryos of P. vancouverensis. However, these data showed that the difference in developmental rate between the two species decreased in succeeding larval stages. There may be convergent selective pressures that result in similar timing to metamorphic competence among phoronid and brachiopod planktotrophic larval types. Morphological differences between these larval types result from heterochronic developmental shifts in the differentiation of juvenile tissue. Similarities in the larval morphology of phoronids and basal deuterostomes are likely the result of functional and developmental constraints rather than a shared (recent) evolutionary origin. These constraints are imposed by the functional design of embryological stages, feeding structures, and swimming structures., (Copyright 2004 Wiley-Liss, Inc.)

A comparative analysis of the larval and presumptive juvenile neuromuscular systems among actinotroch larvae was performed using confocal laser microscopy with probes for F-actin and serotonin. Currently, there are two main categories of larval nervous systems based on the origin of the nerve fibers that innervate the larval tentacles. Characteristics of the serotonergic cells of the larval apical ganglion and juvenile nervous system have remained relatively conserved, but the structure of the secondary (hood) sense organ and the juvenile tentacles has diversified among species. Differences in larval musculature are mainly associated with differences in hood morphology. The presumptive, juvenile neuromuscular system is either integrated or separated from that of the larva based on the origin of the juvenile tentacles. Among species, the juvenile tentacles are made by remodeling the larval tentacles, developed from a basal tentacular thickening, or developed as a completely separate set in the larva. Differentiation of the neuromuscular structures of the juvenile tentacles is more diverse than their outward morphological characteristics would suggest. Importance of these larval characters is discussed in terms of current problems that exist within phoronid systematics. Evolutionary implications of these morphological characters are discussed among the phoronids, brachiopods, and related bilaterians. Overall, the integration or separation of larval and juvenile neuromuscular characters may yield insights into the evolution of lophotrochozoan body plans.

The structure of the larval nervous system and the musculature of Phoronis pallida were studied, as well as the remodeling of these systems at metamorphosis. The serotonergic portion of the apical ganglion is a U-shaped field of cell bodies that send projections into a central neuropil. The majority of the serotonergic cells are (at least) bipolar sensory cells, and a few are nonsensory cells. Catecholaminergic cell bodies border the apical ganglion. The second (hood) sense organ develops at competence and is composed of bipolar sensory cells that send projections into a secondary neuropil. Musculature of the competent larva includes circular and longitudinal muscle fibers of the body wall, as well as elevators and depressors of the tentacles and hood. The juvenile nervous system and musculature are developed prior to metamorphosis and are integrated with those of the larva. Components of the juvenile nervous system include a diffuse neural net of serotonergic cell bodies and fibers and longitudinal catecholaminergic fibers. The juvenile body wall musculature consists of longitudinal fibers that overlie circular muscle fibers, except in the cincture regions, where this pattern is reversed. Metamorphosis is initiated by the larval neuromuscular system but is completed by the juvenile neuromuscular system. During metamorphosis, the larval nervous system and the musculature undergo cell death, and the larval tentacles and gut are remodeled into the juvenile arrangement. Although the phoronid nervous system has often been described as deuterostome-like, these data show that several cytological aspects of the larval and juvenile neuromuscular systems also have protostome (lophotrochozoan) characteristics.

Dysfunction of the tubby protein results in maturity-onset obesity in mice. Tubby has been implicated as a transcription regulator, but details of the molecular mechanism underlying its function remain unclear. Here we show that tubby functions in signal transduction from heterotrimeric GTP-binding protein (G protein)-coupled receptors. Tubby localizes to the plasma membrane by binding phosphatidylinositol 4,5-bisphosphate through its carboxyl terminal "tubby domain." X-ray crystallography reveals the atomic-level basis of this interaction and implicates tubby domains as phosphorylated-phosphatidyl- inositol binding factors. Receptor-mediated activation of G protein alphaq (Galphaq) releases tubby from the plasma membrane through the action of phospholipase C-beta, triggering translocation of tubby to the cell nucleus. The localization of tubby-like protein 3 (TULP3) is similarly regulated. These data suggest that tubby proteins function as membrane-bound transcription regulators that translocate to the nucleus in response to phosphoinositide hydrolysis, providing a direct link between G-protein signaling and the regulation of gene expression.

FokI is a bipartite restriction endonuclease that recognizes a non-palindromic DNA sequence, and then makes double-stranded cuts outside of that sequence to leave a 5' overhang. Earlier kinetic and crystallographic studies suggested that FokI might function as a dimer. Here, we show, using dynamic light-scattering, gel-filtration and analytical ultracentrifugation, that FokI dimerizes only in the presence of divalent metal ions. Furthermore, analysis of the DNA-bound complex reveals that two copies of the recognition sequence are incorporated into the dimeric complex and that formation of this complex is essential for full activation of cleavage. These results have broad implications for the mechanism by which monomeric type II endonucleases achieve high fidelity.

Expression of T-cell receptor and surface immunoglobulins on T and B lymphocytes, respectively, is strictly dependent on the variable, (diversity) joining exon (V(D)J) recombination process, which is initiated by the lymphoid-specific recombinase activating gene proteins 1 and 2 (RAG1 and RAG2). Recent advances have highlighted the functional organization of the RAG1 and RAG2 proteins and have provided important information on the regulation of RAG gene expression. Depending on the severity of their effects on the V(D)J recombination process, mutations of the RAG genes account for a spectrum of combined immune deficiencies in humans.

Severe combined immunodeficiency (SCID) comprises a heterogeneous group of primary immunodeficiencies, a proportion of which are due to mutations in either of the 2 recombination activating genes (RAG)-1 and -2, which mediate the process of V(D)J recombination leading to the assembly of antigen receptor genes. It is reported here that the clinical and immunologic phenotypes of patients bearing mutations in RAGs are more diverse than previously thought and that this variability is related, in part, to the specific type of RAG mutation. By analyzing 44 such patients from 41 families, the following conclusions were reached: (1) null mutations on both alleles lead to the T-B-SCID phenotype; (2) patients manifesting classic Omenn syndrome (OS) have missense mutations on at least one allele and maintain partial V(D)J recombination activity, which accounts for the generation of residual, oligoclonal T-lymphocytes; (3) in a third group of patients, findings were only partially compatible with OS, and these patients, who also carried at least one missense mutation, may be considered to have atypical SCID/OS; (4) patients with engraftment of maternal T cells as a complication of a transplacental transfusion represented a fourth group, and these patients, who often presented with a clinical phenotype mimicking OS, may be observed regardless of the type of RAG gene mutation. Analysis of the RAG genes by direct sequencing is an effective way to provide accurate diagnosis of RAG-deficient as opposed to RAG-independent V(D)J recombination defects, a distinction that cannot be made based on clinical and immunologic phenotype alone.

Omenn's syndrome is an autosomal recessive primary immunodeficiency characterized by variable numbers of T lymphocytes of limited clonality, hypereosinophilia, and high IgE levels with a paradoxical absence of circulating B lymphocytes. We have previously attributed this disorder to missense mutations that render the RAG1/RAG2 recombinase only partially active. Here we report seven Omenn's patients with a novel class of genetic lesions: frameshift mutations within the 5' coding region of RAG1. Interestingly, we demonstrate in transient expression experiments that these frameshift deletion alleles remain partially functional for both deletional and inversional recombination and can hence explain the partial rearrangement phenotype observed in these patients. The rearrangement activity is mediated by truncated RAG1 proteins that are generated by alternative ATG usage 3' to the frameshift deletion and that demonstrate improper cellular localization. Taken together, our results suggest a novel mechanism for the development of immunodeficiency in a subset of Omenn's syndrome patients.

Omenn syndrome (OS) is a peculiar, autosomal recessive severe combined immunodeficiency (SCID) associated with early-onset, generalized, exudative erythrodermia; lymphoadenopathy; hepato- and splenomegaly; hypereosinophilia; elevated serum IgE; and normal to high activated, yet non-functional, oligoclonal T cells. Recent investigations have shown that the primum movens of all these puzzling features lies in a defect of the lymphoid-specific V(D)J recombination process. Abnormalities in both alleles of either Rag-1 or -2 genes are found in all OS patients. At variance with T B- SCID, whose Rag mutations represent null alleles, OS mutations maintain a residual recombination activity, allowing limited T-cell receptor gene rearrangements to occur in the thymus. The gene rearrangements are subsequently expanded in the periphery after environmental antigen exposure. Missense mutations detected in OS have been examined in a number of biochemical assays and have contributed to dissect the various functional domains of both Rag-1 and Rag-2 proteins. The examination of a set of mutations occurring in the Rag-1 N-terminal portion has demonstrated that this region plays a fundamental role in vivo. The elucidation of the molecular basis of OS has allowed us to perform early prenatal diagnosis and could be the basis for trials of in utero bone marrow transplantation or gene therapy approaches.

The V(D)J recombination reaction is composed of multiple nucleolytic processing steps mediated by the recombination-activating proteins RAG1 and RAG2. Sequence analysis has suggested that RAG2 contains six kelch repeat motifs that are predicted to form a six-bladed beta-propeller structure, with the second beta-strand of each repeat demonstrating marked conservation both within and between kelch repeat-containing proteins. Here we demonstrate that mutations G95R and DeltaI273 within the predicted second beta-strand of repeats 2 and 5 of RAG2 lead to immunodeficiency in patients P1 and P2. Green fluorescent protein fusions with the mutant proteins reveal appropriate localization to the nucleus. However, both mutations reduce the capacity of RAG2 to interact with RAG1 and block recombination signal cleavage, therefore implicating a defect in the early steps of the recombination reaction as the basis of the clinical phenotype. The present experiments, performed with an extensive panel of site-directed mutations within each of the six kelch motifs, further support the critical role of both hydrophobic and glycine-rich regions within the second beta-strand for RAG1-RAG2 interaction and recombination signal recognition and cleavage. In contrast, multiple mutations within the variable-loop regions of the kelch repeats had either mild or no effects on RAG1-RAG2 interaction and hence on the ability to mediate recombination. In all, the data demonstrate a critical role of the RAG2 kelch repeats for V(D)J recombination and highlight the importance of the conserved elements of the kelch motif.

During V(D)J recombination, recognition and cleavage of the recombination signal sequences (RSSs) requires the coordinated action of the recombination-activating genes 1 and 2 (RAG1/RAG2) recombinase complex. In this report, we use deletion mapping and site-directed mutagenesis to determine the minimal domains critical for interaction between RAG1 and RAG2. We define the active core of RAG2 required for RSS cleavage as aa 1-371 and demonstrate that the C-terminal 57 aa of this core provide a dominant surface for RAG1 interaction. This region corresponds to the last of six predicted kelch repeat motifs that have been proposed by sequence analysis to fold RAG2 into a six-bladed beta-propeller structure. Residue W317 within this sixth repeat is shown to be critical for mediating contact with RAG1 and concurrently for stabilizing binding and directing cleavage of the RSS. We also show that zinc finger B (aa 727-750) of RAG1 provides a dominant interaction domain for recruiting RAG2. In all, the data support a model of RAG2 as a multimodular protein that utilizes one of its six faces for establishing productive contacts with RAG1.

Tubby-like proteins (TULPs) are found in a broad range of multicellular organisms. In mammals, genetic mutation of tubby or other TULPs can result in one or more of three disease phenotypes: obesity (from which the name "tubby" is derived), retinal degeneration, and hearing loss. These disease phenotypes indicate a vital role for tubby proteins; however, no biochemical function has yet been ascribed to any member of this protein family. A structure-directed approach was employed to investigate the biological function of these proteins. The crystal structure of the core domain from mouse tubby was determined at a resolution of 1.9 angstroms. From primarily structural clues, experiments were devised, the results of which suggest that TULPs are a unique family of bipartite transcription factors.

During V(D)J recombination, processing of branched coding end intermediates is essential for generating junctional diversity. Here, we report that the RAG1/ RAG2 recombinase is a 3' flap endonuclease. Substrates of this nuclease activity include various coding end intermediates, suggesting a direct role for RAG1/ RAG2 in generating junctional diversity during V(D)J recombination. Evidence is also provided indicating that site-specific RSS nicking involves RAG1/RAG2-mediated processing of a localized flap-like structure, implying 3' flap nicking in multiple DNA processing reactions. We have also demonstrated that the bacterial transposase Tn10 contains a 3' flap endonuclease activity, suggesting a mechanistic parallel between RAG1/RAG2 and other transposases. Based on these data, we propose that numerous transposases may facilitate genomic evolution by removing single-stranded extensions during the processing of excision site junctions.

V(D)J recombination is initiated by the specific binding of the RAG1-RAG2 (RAG1/2) complex to the heptamer-nonamer recombination signal sequences (RSS). Several steps of the V(D)J recombination reaction can be reconstituted in vitro with only RAG1/2 plus the high-mobility-group protein HMG1 or HMG2. Here we show that the RAG1 homeodomain directly interacts with both HMG boxes of HMG1 and HMG2 (HMG1,2). This interaction facilitates the binding of RAG1/2 to the RSS, mainly by promoting high-affinity binding to the nonamer motif. Using circular-permutation assays, we found that the RAG1/2 complex bends the RSS DNA between the heptamer and nonamer motifs. HMG1,2 significantly enhance the binding and bending of the 23RSS but are not essential for the formation of a bent DNA intermediate on the 12RSS. A transient increase of HMG1,2 concentration in transfected cells increases the production of the final V(D)J recombinants in vivo.

In this paper, we present the molecular cloning and characterization of a murine homolog of the Escherichia coli chaperone ClpX. Murine ClpX shares 38% amino acid sequence identity with the E. coli homolog and is a novel member of the Hsp100/Clp family of molecular chaperones. ClpX localizes to human chromosome 15q22.2-22.3 and in mouse is expressed tissue-specifically as one transcript of approximately 2.9 kilobases (kb) predominantly within the liver and as two isoforms of approximately 2.6 and approximately 2.9 kb within the testes. Purified recombinant ClpX displays intrinsic ATPase activity, with a Km of approximately 25 microM and a Vmax of approximately 660 pmol min-1 microgram-1, which is active over a broad range of pH, temperature, ethanol, and salt parameters. Substitution of lysine 300 with alanine in the ATPase domain P-loop abolishes both ATP hydrolysis and binding. Recombinant ClpX can also interact with its putative partner protease subunit ClpP in overexpression experiments in 293T cells. Subcellular studies by confocal laser scanning microscopy localized murine ClpX green fluorescent protein fusions to the mitochondria. Deletion of the N-terminal mitochondrial targeting sequence abolished mitochondrial compartmentalization. Our results thus suggest that murine ClpX acts as a tissue-specific mammalian mitochondrial chaperone that may play a role in mitochondrial protein homeostasis.

In vertebrates, generation of the T- and B-cell repertoire relies on genomic rearrangement of T-cell receptor and immunoglobulin gene coding segments. This process, known as V(D)J recombination, is initiated by the lymphoid specific proteins Rag1 and Rag2. Both in humans and in animal models, mutations that abrogate expression of either the Rag1 or Rag2 proteins result in severe combined immune deficiency with a complete lack of circulating T and B cells due to an early block in lymphoid development. We have recently shown that mutations that impair, but do not completely abolish the function of Rag1 and Rag2 in humans result in Omenn syndrome, an enigmatic form of combined immune deficiency characterized by oligoclonal, activated T lymphocytes with a skewed Th2 profile.

V(D)J site-specific recombination mediates the somatic assembly of the antigen receptor gene segments. This process is initiated by the recombination activating proteins RAG1 and RAG2, which recognize the recombination signal sequences (RSS) and cleave the DNA at the coding/RSS junction. In this study, we show that RAG1 and RAG2 have the ability to directly interact in solution before binding to the DNA. RAG1 forms a homodimer, which leads to the appearance of two distinct RAG1.RAG2 complexes bound to DNA. To investigate the properties of the two RAG1.RAG2 complexes in the presence of different Me2+ cofactors, we established an in vitro Mg2+-based cleavage reaction on a single RSS. Using this system, we found that Mg2+ confers a specific pattern of DNA binding and cleavage. In contrast, Mn2+ allows aberrant binding of RAG1.RAG2 to single-stranded RSS and permits cleavage independent of binding to the nonamer. To determine the contribution of Me2+ ions at the early stages of V(D)J recombination, we analyzed specific DNA recognition and cleavage by RAG1.RAG2 on phosphorothioated substrates. These experiments revealed that Me2+ ions directly coordinate the binding of RAG1.RAG2 to the RSS DNA.

Genomic rearrangement of the antigen receptor loci is initiated by the two lymphoid-specific proteins Rag-1 and Rag-2. Null mutations in either of the two proteins abrogate initiation of V(D)J recombination and cause severe combined immunodeficiency with complete absence of mature B and T lymphocytes. We report here that patients with Omenn syndrome, a severe immunodeficiency characterized by the presence of activated, anergic, oligoclonal T cells, hypereosinophilia, and high IgE levels, bear missense mutations in either the Rag-1 or Rag-2 genes that result in partial activity of the two proteins. Two of the amino acid substitutions map within the Rag-1 homeodomain and decrease DNA binding activity, while three others lower the efficiency of Rag-1/Rag-2 interaction. These findings provide evidence to indicate that the immunodeficiency manifested in patients with Omenn syndrome arises from mutations that decrease the efficiency of V(D)J recombination.

The V(D)J recombinase subunits Rag-1 and Rag-2 mediate assembly of antigen receptor gene segments. We studied the mechanisms of DNA recognition by Rag-1/Rag-2 using surface plasmon resonance. The critical step for signal recognition is binding of Rag-1 to the nonamer. This is achieved by a region of Rag-1 homologous to the DNA-binding domain of the Hin family of bacterial invertases and to homeodomain proteins. Strikingly, the Hin homeodomain can functionally substitute for the Rag-1 homologous region. Rag-1 also interacts with the heptamer but with low affinity. Rag-2 shows no direct binding to DNA. Once the Rag-1/Rag-2 complex is engaged on the DNA, subsequent cleavage is directed by the heptamer sequence. This order of events remarkably parallels mechanisms that mediate transposition in bacteria and nematodes.