Genotoxic aldehydes in the hematopoietic system.

Reactive aldehydes are potent genotoxins that threaten the integrity of hematopoietic stem cells and blood production. To protect against aldehydes, mammals have evolved a family of enzymes to detoxify aldehydes, and the Fanconi anemia DNA repair pathway to process aldehyde-induced DNA damage. Loss of either protection mechanisms in humans results in defective hematopoiesis and predisposition to leukemia. This review will focus on the impact of genotoxic aldehydes on hematopoiesis, the sources of endogenous aldehydes, and potential novel protective pathways.

Why does the bone marrow fail in Fanconi anemia?

The inherited bone marrow failure (BMF) syndromes are a rare and diverse group of genetic disorders that ultimately result in the loss of blood production. The molecular defects underlying many of these conditions have been elucidated, and great progress has been made toward understanding the normal function of these gene products. This review will focus on perhaps the most well-known and genetically heterogeneous BMF syndrome: Fanconi anemia. More specifically, this account will review the current state of our knowledge on why the bone marrow fails in this illness and what this might tell us about the maintenance of bone marrow function and hematopoiesis.

FANCJ coordinates two pathways that maintain epigenetic stability at G-quadruplex DNA.

We have previously reported that DT40 cells deficient in the Y-family polymerase REV1 are defective in replicating G-quadruplex DNA. In vivo this leads to uncoupling of DNA synthesis from redeposition of histones displaced ahead of the replication fork, which in turn leads to loss of transcriptional repression due to failure to recycle pre-existing repressive histone post-translational modifications. Here we report that a similar process can also affect transcriptionally active genes, leading to their deactivation. We use this finding to develop an assay based on loss of expression of a cell surface marker to monitor epigenetic instability at the level of single cells. This assay allows us to demonstrate G4 DNA motif-associated epigenetic instability in mutants of three helicases previously implicated in the unwinding of G-quadruplex structures, FANCJ, WRN and BLM. Transcriptional profiling of DT40 mutants reveals that FANCJ coordinates two independent mechanisms to maintain epigenetic stability near G4 DNA motifs that are dependent on either REV1 or on the WRN and BLM helicases, suggesting a model in which efficient in vivo replication of G-quadruplexes often requires the established 5'-3'-helicase activity of FANCJ acting in concert with either a specialized polymerase or helicase operating in the opposite polarity.

Monoubiquitylation in the Fanconi anemia DNA damage response pathway.

The hereditary genetic disorder Fanconi anemia (FA) belongs to the heterogeneous group of diseases associated with defective DNA damage repair. Recently, several reviews have discussed the FA pathway and its molecular players in the context of genome maintenance and tumor suppression mechanisms [H. Joenje, K.J. Patel, The emerging genetic and molecular basis of Fanconi anaemia, Nat. Rev. Genet. 2 (2001) 446-457; W. Wang, Emergence of a DNA-damage response network consisting of Fanconi anaemia and BRCA proteins, Nat. Rev. Genet. 8 (2007) 735-748; L.J. Niedernhofer, A.S. Lalai, J.H. Hoeijmakers, Fanconi anemia (cross)linked to DNA repair, Cell 123 (2005) 1191-1198; K.J. Patel, Fanconi anemia and breast cancer susceptibility, Nat. Genet. 39 (2007) 142-143]. This review assesses the influence of post-translational modification by ubiquitin. We review and extract the key features of the enzymatic cascade required for the monoubiquitylation of the FANCD2/FANCI complex and attempt to include recent findings into a coherent mechanism. As this part of the FA pathway is still far from fully understood, we raise several points that must be addressed in future studies.

Microarrays: applications in dental research.

Revolutionary advances are underway that will dramatically change our understanding of oral diseases. The phenomenal progress being made in biomedical research is in large part fueled by advances in our overall knowledge of the human genome, development of microarray technology that allows comprehensive and unbiased evaluation of global biologic pathways and networks, and expanded computational abilities. Expectations are that nearly all clinical areas in dentistry and oral medicine will be affected by advances in molecular medicine, which in turn, promises to lead to more accurate diagnosis, effective disease monitoring, and development of targeted and specific therapies. This review provides a brief overview of microarray technologies and highlights several key examples from research efforts in dentistry and oral medicine using these powerful new tools.

Evaluation of the prevalence and economic burden of adverse drug reactions presenting to the medical emergency department of a tertiary referral centre: a prospective study.

BACKGROUND: Adverse drug reactions (ADRs) are now recognized as an important cause of hospital admissions, with a proportion ranging from 0.9-7.9%. They also constitute a significant economic burden. We thus aimed at determining the prevalence and the economic burden of ADRs presenting to Medical Emergency Department (ED) of a tertiary referral center in India METHODS: A prospective, observational study of adult patients carried out over a 6 week period in 2005. The prevalence of ADRs, their economic burden from the hospital perspective, severity, and preventability were assessed using standard criteria. RESULTS: A total 6899 patients presented during the study period. Of these, 2046 were admitted for various reasons. A total of 265/6899 patients had ADRs (3.84 %). A total of 141/265 was admitted due to ADsR, and thus ADRs as a cause of admissions were 6.89% of total admissions. A majority (74.71%) were found to be of moderate severity. The most common ADRs were anti-tubercular drug induced hepatotoxicity, warfarin toxicity and chloroquine induced gastritis. The median duration of hospitalization was 5 days [95% CI 5.37, 7.11], and the average hospitalization cost incurred per patient was INR 6197/- (USD 150). Of total ADRs, 59.62% (158/265) were found to be either definitely or potentially avoidable. CONCLUSION: The study shows that ADRs leading to hospitalization are frequent and constitute a significant economic burden. Training of patients and prescribers may lead to a reduction in hospitalization due to avoidable ADRs and thus lessen their economic burden.

The Fanconi anemia gene product FANCF is a flexible adaptor protein.

The Fanconi anemia (FA) protein FANCF is an essential component of a nuclear core complex that protects the genome against chromosomal instability, but the specific function of FANCF is still poorly understood. Based upon the homology between human and Xenopus laevis FANCF, we carried out an extensive mutagenesis study to examine which domains are functionally important and to gain more insight into the function of FANCF. In contrast to previous suggestions, we show that FANCF does not have a ROM-like function. We found that the C terminus of FANCF interacts directly with FANCG and allows the assembly of other FA proteins into a stable complex. The N terminus appears to stabilize the interaction with FANCA and FANCG and is essential for the binding of the FANCC/FANCE subcomplex. We identified several important amino acids in this N-terminal region but, surprisingly, many amino acid changes failed to affect the function of the FANCF protein. Our data demonstrate that FANCF acts as a flexible adaptor protein that plays a key role in the proper assembly of the FA core complex.

FANCE: the link between Fanconi anaemia complex assembly and activity.

The Fanconi anaemia (FA) nuclear complex (composed of the FA proteins A, C, G and F) is essential for protection against chromosome breakage. It activates the downstream protein FANCD2 by monoubiquitylation; this then forges an association with the BRCA1 protein at sites of DNA damage. Here we show that the recently identified FANCE protein is part of this nuclear complex, binding both FANCC and FANCD2. Indeed, FANCE is required for the nuclear accumulation of FANCC and provides a critical bridge between the FA complex and FANCD2. Disease-associated FANCC mutants do not bind to FANCE, cannot accumulate in the nucleus and are unable to prevent chromosome breakage.

The emerging genetic and molecular basis of Fanconi anaemia.

The past few years have witnessed a considerable expansion in our understanding of the pathways that maintain chromosome stability in dividing cells through the identification of genes that are mutated in certain human chromosome instability disorders. Cells that are derived from patients with Fanconi anaemia (FA) show spontaneous chromosomal instability and mutagen hypersensitivity, but FA poses a unique challenge as the nature of the DNA-damage-response pathway thought to be affected by the disease has long been a mystery. However, the recent cloning of most of the FA-associated genes, and the characterization of their protein products, has provided tantalizing clues as to the molecular basis of this disease.

A recombinant rat regenerating protein is mitogenic to pancreatic derived cells.

Pancreatic regenerating protein (reg I) is expressed in acinar cells and is mitogenic to beta- and ductal cells. Isolation of large amounts of endogenous reg I for in vivo and in vitro experiments has been difficult. The aim of this study was to develop a recombinant protein and determine its bioactivity on rat pancreatic derived cells. cDNA of the rat reg I coding region was created with unique BamHI flanking sequences using reverse transcriptase PCR. The coding region was then cloned into a bacterial expression vector in which expression is controlled by a T7 promoter. After transformation into the Escherichia coli strain B21(DE3) and induction by isopropyl-beta-d-thiogalactopyranoside, a fusion protein of 24 kDa in size, named reg-PET, was noted in the bacterial lysate. This protein contained a polyhistidine and S-peptide sequence to facilitate isolation and identification, respectively. This protein was purified using affinity chromatography, and identity was confirmed with gel electrophoresis and Western analysis. The reg-PET protein was mitogenic to both ARIP and RIN cells, rat pancreatic ductal and beta-cell lines, respectively. Antibodies raised to the protein reacted against rat reg I in pancreas. The purified recombinant reg I fusion protein, like endogenous reg I, is mitogenic to pancreatic derived cells. It is more potent than reg I isolated from pancreatic tissue. This protein can be isolated rapidly, easily, and with a high amount of purity.

Involvement of Brca2 in DNA repair.

Abnormalities precipitated by a targeted truncation in the murine gene Brca2 define its involvement in DNA repair. In culture, cells harboring truncated Brca2 exhibit a proliferative impediment that worsens with successive passages. Arrest in the G1 and G2/M phases is accompanied by elevated p53 and p21 expression. Increased sensitivity to genotoxic agents, particularly ultraviolet light and methylmethanesulfonate, shows that Brca2 function is essential for the ability to survive DNA damage. But checkpoint activation and apoptotic mechanisms are largely unaffected, thereby implicating Brca2 in repair. This is substantiated by the spontaneous accumulation of chromosomal abnormalities, including breaks and aberrant chromatid exchanges. These findings define a function of Brca2 in DNA repair, whose loss precipitates replicative failure, mutagen sensitivity, and genetic instability reminiscent of Bloom syndrome and Fanconi anemia.

The nature of ligand-induced conformational change in transferrin in solution. An investigation using X-ray scattering, XAFS and site-directed mutants.

Ligand-induced conformational change in transferrins has been studied by site-directed mutagenesis of human serum half molecule (N-lobe), X-ray absorption fine structure (XAFS) spectroscopy and X-ray solution scattering. Use of recent advances in data analysis has been made for extracting model-independent molecular shapes from X-ray solution scattering data for the intact, the half molecule and its mutants. Clear evidence is provided that the transferrin molecule (intact as well as N-lobe), in its apo and holo forms, exists for the majority of the time in well-defined specific conformations representing the "fully opened" and "closed" states of the molecule, respectively. Evidence is also provided for the existence of an additional conformation, referred to here as the "intermediate" conformation for simplicity, which is trapped in the case of some of the mutants in the iron-bound form. We suggest that domain closure in the transferrin molecule is a two-step process, with the intermediate conformation representing the first stage of domain closure (approximately 20 degrees hinge-twist of domain II). Our data are not inconsistent with the ligand-free molecule sampling the closed states occasionally (< or = 10%) but are not in support of a continuous conformational search between the fully opened and closed states in the absence of iron.

Thymic lymphomas in mice with a truncating mutation in Brca2.

Inherited mutations in the BRCA2 gene predispose women to breast and ovarian cancer. We created a mutation in the mouse Brca2 gene that terminates translation in exon 11 at 45% of the normal transcript length. Ninety % of Brca2(tm1Cam) homozygous mutant mice die prenatally or perinatally. The location of the Brca2(tm1Cam) mutation differs from those reported previously, and this phenotype suggests a correlation with genotype analogous to that previously reported in humans. Although heterozygote mice have remained free of tumors for 10 months, Brca2(tm1Cam) homozygous mutants that survived to adulthood died with thymic lymphomas between 12 and 14 weeks of age.

Milwaukee shoulder with massive bilateral cysts: effective therapy for hydrops of the shoulder.

"Milwaukee shoulder" is often associated with large effusions that cannot be managed with conventional therapy. We describe a 75-year-old man whose massive hydrops of both shoulders was resistant to treatment with nonsteroidal antiinflammatory drugs (NSAID), multiple aspirations, and injections of corticosteroid. The effusions resolved completely after treatment with oral colchicine and an NSAID containing magnesium.

Iron release from recombinant N-lobe and mutants of human transferrin.

Mutations of kinetically active residues in the recombinant N-lobe of human transferrin may accelerate or retard release of iron from the protein to pyrophosphate, thereby providing means for exploring the individual roles of such residues in the concerted mechanisms of release. Using an established spectrofluorometric method and pyrophosphate as the required iron-sequestering agent, we have compared release from unaltered native transferrin and recombinant N-lobe half-transferrin to release from six N-lobe mutants, R124S, R124K, K206R, H207E, H249Y, and Y95H. Mutation of R124, which serves as a principal anchor for the synergistic carbonate anion ordinarily required for iron binding by transferrin, accelerates release. This effect is most marked at endosomal pH, 5.6, and is also evident at extracellular pH, 7.4, pointing to a critical and perhaps initiating role of carbonate in the release process. Mutation of K206 to arginine, or of H207 to glutamine, each lying in the interdomain cleft of the N-lobe, gives products mimicking the arrangements in lactoferrin. Release of iron from these two mutants, as from lactoferrin, is substantially slower than from unaltered recombinant N-lobe. Interdomain residues not directly involved in iron or anion binding may therefore participate in the control of iron release within the endosome. The H249Y mutant releases iron much more rapidly than its wild-type parent or any other mutant, possibly because of steric effects of the additional phenolic ring in the binding site. No simple explanation is available to account for a stabilizing effect of the Y95H mutation.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization and structural analysis of a functional human serum transferrin variant and implications for receptor recognition.

The nucleotide and amino acid substitutions leading to the only known functional variant of human serum transferrin have been characterized by sequencing of a peptide produced by cyanogen bromide digestion and genomic PCR coupled with cycle sequencing, respectively. There is an amino acid substitution at position 394 (Gly-->Arg) resulting from a mutational transition, G-->A, in the first nucleotide of the codon GGG. The Zn(2+)-, Al(3+)-, and Cu(2+)-binding properties of the variant, ascertained by UV difference spectra and, in the case of copper, protein fluorescence quenching, confirm that these metals binds to only one of the two sites. Solution X-ray scattering measurements indicate that the lobe (the C-lobe) containing the mutation remains "open" in the iron-bound state, and modeling studies suggest that this is a consequence of the formation of a salt bridge between Arg394 in the variant protein and Asp392, one of the iron-binding ligands in the C-lobe. This rationalizes for the first time the observed reduction in receptor affinity of the diferric variant protein for PHA-stimulated lymphocytes [Young, S.P., et al. (1984) Br. J. Haematol. 56, 581-587] and here repeated with K562 cells. These data lend support to the hypothesis that the closed conformation for both lobes contributes to receptor recognition.

Evidence for the glycosylation of porcine serum transferrin at a single site located within the C-terminal lobe.

In this study we report the number and location of the glycans on PST. Urea PAGE and SDS-PAGE have been used to follow the enzymatic removal of sialic acids and of glycans from PST and the masses of native and deglycosylated PST have been determined by electrospray mass spectrometry. The results are consistent with the presence of a single biantennary glycan chain. As amino acid sequence analysis demonstrated the absence of a glycosylated asparagine at position 25, the glycosylation site is restricted to Asp-497.

The alpha/beta sheath and its cytoplasmic tyrosines are required for signaling by the B-cell antigen receptor but not for capping or for serine/threonine-kinase recruitment.

The B-cell antigen receptor is composed of membrane immunoglobulin sheathed by an alpha/beta heterodimer. The complex is noncovalently associated with protein kinase activity, and crosslinking of the receptor leads to capping and transmembrane signaling. Here we show that the sheath is not necessary either for this capping or for the association of membrane immunoglobulin with the detergent-insoluble cytoskeletal fraction that occurs following crosslinking. It is also not required for association of membrane immunoglobulin with a casein-kinase-like serine/threonine kinase. The sheath is essential, however, for transmembrane signaling. Provision of just the cytoplasmic domain of the beta sheath polypeptide to a mutant, unsheathed IgM molecule was sufficient to restore full signaling capability as judged by the phosphorylation of a variety of cellular proteins, including the B-cell-specific transmembrane protein CD22. This signaling was destroyed by mutating one of the tyrosines in the beta cytoplasmic domain. These results not only suggest that receptor signaling is mediated through phosphorylation of the tyrosines in the sheath's cytoplasmic domains but, together with previous work, indicate that different motifs within the sheath mediate presentation and signaling.

Antigen presentation by the B cell antigen receptor is driven by the alpha/beta sheath and occurs independently of its cytoplasmic tyrosines.

Membrane immunoglobulin functions to internalize bound antigen for its subsequent processing and presentation to T cells. Although the five immunoglobulin isotypes exhibit considerable differences in their cytoplasmic domains, we show by use of matched B lymphoma transfectants that all isotypes manifest a similar high efficacy in antigen presentation. Experiments using mutant receptors reveal that this efficacy can be ascribed to the alpha/beta sheath of the receptor, where presentation correlates with internalization of polyvalent antigen. Efficient presentation is restored to a sheathless antigen receptor by providing it with only the cytoplasmic domain of the beta sheath polypeptide. This restoration of activity does not depend on the tyrosine residues in the beta cytoplasmic tail, implying that antigen receptor-mediated presentation can occur by a pathway distinct from that used by the Fc receptor Fc gamma RIII.

The mouse B-cell antigen receptor: definition and assembly of the core receptor of the five immunoglobulin isotypes.

We have shown that the core antigen receptor of all five isotypes is composed of immunoglobulin in association with a common heterodimeric alpha/beta sheath. The stoichiometry of the association is unknown although preliminary evidence points to it being an IgH2L2 [alpha/beta]2 association. Studies with chimaeric molecules indicate that much of the immunoglobulin-sheath interaction must occur through the carboxyterminal end of the molecule with particular importance being given to the linker-transmembrane region. The glycosylation of the alpha chain differs according to the isotype with which it is associated. There are two sites for N-linked glycosylation on the alpha chain (Asn-30 and Asn-40); both sites are used. Mutation of Asn-30 alone decreases but does not abolish surface expression of the antigen receptor complex. Mutation of both sites prevents expression of the surface IgM[alpha/beta] complex but not of a surface IgD[alpha/beta] complex. Moreover, the pattern of alpha glycosylation is considerably affected by changes in the linker region between C mu 4 and the transmembrane, giving further support to the importance of this region in immunoglobulin-sheath interaction. Unlike IgM, IgD and IgG2b do not require alpha/beta for transport to the cell surface and can be expressed on the surface without either sheath or glycosyl phosphatidylinositol anchor. This finding may reflect that the IgD transmembrane region is significantly less hydrophobic than that of IgM; however, it should be noted that is not clear whether naked IgD exists in vivo. In fact, we have found that the alpha/beta sheath is necessary in order to facilitate efficient internalization and presentation of antigen by membrane immunoglobulin. The sheath presumably also plays a major role in potentiating transmembrane signalling. However, mutant receptors that do not associate with the alpha/beta sheath are nevertheless able to trigger phosphorylation of cellular proteins on tyrosine residues following cross-linking. Also, in addition to the alpha/beta sheath, other transmembrane proteins associate with the B-cell antigen receptor although they are not required in order to potentiate surface transport. It may be interaction with one of these other associated transmembrane proteins or, alternatively, interaction between the immunoglobulin cytoplasmic tail and, say, the cytoskeleton, that enables antigen receptors lacking the alpha/beta sheath to give rise to cellular signalling.