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.

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.

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.

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 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.

Evidence for a single glycan moiety in rabbit serum transferrin and location of the glycan within the polypeptide chain.

The sequential removal of N-acetylneuraminic acid from rabbit serum transferrin has been followed by urea-polyacrylamide gel electrophoresis. The electrophoretic pattern is consistent with the presence of a single biantennary glycan chain. From the amino acid sequence of the carbohydrate-containing cyanogen bromide fragment we have shown that the glycan is attached to an asparaginyl side chain at a position equivalent to residue 491 in the sequence of human serum transferrin.

Electron spin resonance spin label studies of the erythrocyte membrane in Duchenne muscular dystrophy.

The membrane organisation of erythrocytes from patients with Duchenne muscular dystrophy (DMD) and from asymptomatic carriers was studied by the electron spin resonance (ESR) spin label technique. Following the work of Sato et al [1978], we used 2-(14-carboxytetradecyl)-2-ethyl-4, 4-dimethyl-3-oxazolidinyloxyl as probe. We found no significant difference in lipid fluidity at 30 degrees C, measured by the ratio of the line height at central field to that at high field ho/h-1, in DMD patients or carriers compared to appropriate control persons. Our conclusions for the dystrophic boys differ from those of Sato et al [1978], although our data are consistent with theirs within experimental error. We also studied the variation of the ratio ho/h-1 over the temperature range 5-35 degrees C in these individuals. For normal erythrocytes there is a discontinuity in this plot around 15 degrees C that is absent in the erythrocytes of DMD patients [in agreement with the findings of Sato et al, 1978] and also absent in DMD carriers. We suggest the slope of this logarithmic temperature plot over the range 15-35 degrees C is a more useful empirical parameter as the presence of these discontinuities is sometimes uncertain, and using this parameter, we find a clear separation between carriers and controls.

Temperature-dependent development of the fungal pathogen Lagenidium giganteum (Oomycetes: Lagenidiales) in larvae of Culex quinquefasciatus (Diptera: Culicidae).

The rates of development of Lagenidium giganteum were determined in the four larval instars of Culex quinquefasciatus Say held at 15, 20, 25, 27, 30, and 34 degrees C. The fastest development was in second instars held at 34 degrees; vesicles and oospores occurred in 50% of the larvae (the median development time) 19.7 and 25.0 h, respectively, after infection. The greatest median time to the formation of vesicles was in third instars at 15 degrees C (185.6 h) and for oospores was in second instars at 15 degrees C (152.3 h). The fungus did not form oospores in fourth instars at 15 degrees C. The median developmental rates of vesicles and oospores in each instar were fit to the Sharpe & DeMichele model, which may be used to predict the effects of different temperatures on the in-vivo developmental rate of the fungus.

Temperature-dependent development and survival rates of Culex quinquefasciatus and Aedes aegypti (Diptera: Culicidae).

Development, growth, and survival of Culex quinquefasciatus Say and Aedes aegypti (L.) were determined at six constant temperatures (15, 20, 25, 27, 30, 34 degrees C). The Sharpe & DeMichele four-parameter model with high-temperature inhibition described the temperature-dependent median developmental rates of both mosquito species. In both species, body size generally decreased as temperature increased. Head capsule widths in all instars in both species were significantly greater at 15 than at 30-34 degrees C. Except for the third instar of Ae. aegypti, the larval body lengths in both species were significantly greater at 15 than at 34 degrees C. All instars and pupae of both species and the adults in Cx. quinquefasciatus were significantly heavier at 15 than at 27-34 degrees C. In Cx. quinquefasciatus, survival from eclosion to adult emergence was highest in the range from 20 to 30 degrees C (85-90%) and dropped drastically at 15 (38%) and 34 degrees C (42%). In Ae. aegypti, survival to adult stage was high at 20 (92%) and 27 degrees C (90%) and lowest at 15 degrees C (3%).

Comparison of floating and sinking encapsulated formulations of the fungus Lagenidium giganteum (Oomycetes: Lagenidiales) for control of Anopheles larvae.

Floating calcium alginate capsules containing Lagenidium giganteum and 1% ground cork gave higher levels of control of Anopheles quadrimaculatus larvae in a 100-cm column of water than sinking capsules containing no cork. There was no significant difference between the cork capsules and the sinking capsules in the infection of larvae by the encapsulated fungus after storage (15 degrees C) for 57 days, although infectivity declined during that time from an initial infection rate of 100% to 35% and 40% for the cork and sinking capsules, respectively. Floating capsules containing glass bubbles were less effective than the cork capsules in the 100-cm column of water and had a shorter storage life than either sinking capsules or cork capsules.

Comparisons of different types and concentrations of alginates for encapsulation of Lagenidium giganteum (Oomycetes: Lagenidiales), a fungal pathogen of mosquito larvae.

Six different types of alginates used to encapsulate Lagenidium giganteum gave similar levels of fungal infection in Culex quinquefasciatus larvae. Initial infection levels when the capsules were immersed in water after 6 days of storage (15 and 25 degrees C) were 100% for all types of alginate and after 42 days of storage was 62-100%, depending on the type of alginate. Infectivity was 24-100% after the encapsulated fungus were left in water for 7 days and after 15 days was 0 to 26%, depending on the alginate. When 2 of the alginates were tested at different concentrations to give high, medium and low viscosity solutions, the fungus encapsulated using lower concentration alginate solutions usually gave the highest level of infectivity.

Efficacy of encapsulated Lagenidium giganteum (Oomycetes: Lagenidiales) against Culex quinquefasciatus and Aedes aegypti larvae in artificial containers.

Presporangial mycelia of Lagenidium giganteum cultured on sunflower seed extract were encapsulated in calcium alginate and added once (July 18) to outdoor (Raleigh, NC) caged tires, wood and concrete containers populated with first instars of Culex quinquefasciatus or Aedes aegypti. First instars were added twice weekly (for 10 wk) to simulate natural oviposition. The fungus persisted for 10 wk and recycled in the mosquito larvae of both species. The overall reductions of Cx. quinquefasciatus and Ae. aegypti immatures were higher in tires (55 and 45%, respectively) and wood (67 and 38%) than in concrete containers (17 and 14%). There were low correlations of the numbers of mosquito immatures with measurements of water quality (chemical oxygen demand, ammonia nitrogen and conductivity) in the containers.

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.

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 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.