Isolation and phenogenetics of a novel circadian rhythm mutant in zebrafish

Widespread use of zebrafish (Danio rerio) in genetic analysis of embryonic development has led to rapid advances in the technology required to generate, map and clone mutated genes. To identify genes involved in the generation and regulation of vertebrate circadian rhythmicity, we screened for dominant mutations that affect the circadian periodicity of larval zebrafish locomotor behavior. In a screen of 6,500 genomes, we recovered 8 homozygous viable, semi-dominant mutants, and describe one of them here. The circadian period of the lager and lime (lag(dg2)) mutant is shortened by 0.7 h in heterozygotes,and 1.3 h in homozygotes. This mutation also shortens the period of the melatonin production rhythm measured from cultured pineal glands, indicating that the mutant gene product affects circadian rhythmicity at the tissue level, as well as at the behavioral level. This mutation also alters the sensitivity of pineal circadian period to temperature, but does not affect phase shifting responses to light. Linkage mapping with microsatellite markers indicates that the lag mutation is on chromosome 7. A zebrafish homolog of period1(per1) is the only known clock gene homolog that maps near the lag locus. However, all sequence variants found in per1 cDNA from lag(dg2) mutants are also present in wild type lines, and we were unable to detect any defect in per1 mRNA splicing, so this mutation may identify a novel clock gene.     

Autonomously replicating plasmids carrying theAMA1 region inPenicillium chrysogenum

Plasmid vectors containing theAMA1 sequence transformed with high efficiency and replicated autonomously inPenicillium chrysogenum. The efficiency of transformation ofP. chrysogenum was related to the length of theAMA1 fragment used for constructing the different autonomously replicating plasmids. One of the two palindromic inverted repeats ofAMA1 (the 2.2-kbSalI-HindIII fragment) is sufficient to confer autonomous replication and a high transformation efficiency. Deletion of the 0.6-kb central fragment located between the inverted repeats did not affect either the ability of the plasmids to replicate autonomously or the efficiency of transformation, but did alter the mitotic stability and the plasmid copy number. Deletion of any fragment of the 2.2-kb repeat caused the loss of the ability to replicate autonomously and reduced the transformation efficiency. Most of the transformants retained the original plasmid configuration, as multimers and without reorganization, after several rounds of autonomous replication. TheAMA1 region works as an origin of replication inP. chrysogenum andA. nidulans but not apparently inAcremonium chrysogenum.     

Experimental infection of immunomodulated NOD/LtSz-SCID mice as a new model for Plasmodium falciparum erythrocytic stages

The main objective of this study was to determine whether a chemical immunomodulation protocol could reduce the resistance of NOD/LtSz-SCID mice to Plasmodium falciparum infection and provide an improved mouse model for screening the antimalarial activity of new compounds. This model was compared with the presently used immunodeficient Beige/Nude/Xid (BNX) mouse model, using the same protocol, in terms of percentage of infected mice, parasite development, leukocyte response and phagocytosis of P. falciparum infected cells in various organs. Our results show that the combination of the chemical immune modulation protocol with the genetic background of NOD/LtSz-SCID mice results in the development of long-lasting P. falciparum infection in a high percentage of mice. A comparison of the results obtained in the histological study for both mouse models suggests that the higher rate of success in NOD/LtSz-SCID mice could be related to the reduced macrophage recruitment developed in different tissues to remove the parasite from blood.     

Conservation of epitopes recognized by monoclonal antibodies against the separated subunits of influenza hemagglutinin among type A viruses of the same and different subtypes

Summary Monoclonal antibodies raised against the separated hemagglutinin subunits (HA1 and HA2) of influenza A/Vic/3/75 (H3N2) virus were tested against a large panel of human and avian strains. The epitopes recognized by most antibodies were conserved among subtype H3 viruses, but reactivity of some antibodies with members of other subtypes was also observed. Particularly, the H4 virus reacted with most antibodies directed against the HA2 subunit. These results are discussed in terms of sequence similarities between subtypes and application of these antibodies as subtyping reagents.     

Haplotype analysis of the BRCA2 9254delATCAT recurrent mutation in breast/ovarian cancer families from Spain

A frame-shift 9254del5 mutation was independently identified in 12 families, eleven of them with Spanish ancestors, in a BRCA2 screening performed in 841 breast and/or ovarian cancer families and in 339 women with breast cancer diagnosed before the age of 40 at different centers in France and Spain. We sought to analyze in detail the haplotype and founder effects of the 9254del5 and to estimate the time of origin of the mutation. Eight polymorphic microsatellite markers and two BRCA2 polymorphisms were used for the haplotype analyses. The markers were located flanking the BRCA2 gene spanning a region of 6.1 cM. Our results suggest that these families shared a common ancestry with BRCA2 9254del5, which is a founder mutation originating in the Northeast Spanish, with an estimated age of 92 (95% CI 56-141) generations.     

Relative food intake of rats submitted to a moderate transgenerational undernutrition

An experiment on rat undernutrition through seven generations was performed in order to see: (1) whether the nutritional stress on growth increases from one generation to the next, and (2) if an equilibrium point (AFP) in which the RFI--the amount of food intake (mg) per gram of body weight--reached is the same in both control and undernourished animals. The RFI values were calculated for each generation, between the 30th and 100th days of age. A moderate undernutrition was applied to the seven generations (F1 to F7) following the parental (P) one, which acted as controls. Undernourishment was made from conception to the end of the experiment (100 days old). The RFI values diminished with the age increment and increased through generations. There was, however, a clear AFP of 75.9 +/- 3.5 mg/g at 100 days of age in males, and of 78.7 +/- 4.2 mg/g at 90 days of age in females. A clear cumulative increment of RFI through the filial generations was also found at intermediate growth ages. The frequently argued nongenetic transmission of the nutritional deficiencies from parents to descendants was corroborated with the present results. Such cumulative effect was evident at ages before the AFP was reached; i.e., when the decrement in body mass of the undernourished animals was not yet equilibrated with the amount of available nutrients.     

Discriminatory power and reproducibility of novel DNA typing methods for Mycobacterium tuberculosis complex strains

In recent years various novel DNA typing methods have been developed which are faster and easier to perform than the current internationally standardized IS6110 restriction fragment length polymorphism typing method. However, there has been no overview of the utility of these novel typing methods, and it is largely unknown how they compare to previously published methods. In this study, the discriminative power and reproducibility of nine recently described PCR-based typing methods for Mycobacterium tuberculosis were investigated using the strain collection of the interlaboratory study of Kremer et al. This strain collection contains 90 M. tuberculosis complex and 10 non-M. tuberculosis complex mycobacterial strains, as well as 31 duplicated DNA samples to assess reproducibility. The highest reproducibility was found with variable numbers of tandem repeat typing using mycobacterial interspersed repetitive units (MIRU VNTR) and fast ligation-mediated PCR (FLiP), followed by second-generation spoligotyping, ligation-mediated PCR (LM-PCR), VNTR typing using five repeat loci identified at the Queens University of Belfast (QUB VNTR), and the Amadio speciation PCR. Poor reproducibility was associated with fluorescent amplified fragment length polymorphism typing, which was performed in three different laboratories. The methods were ordered from highest discrimination to lowest by the Hunter-Gaston discriminative index as follows: QUB VNTR typing, MIRU VNTR typing, FLiP, LM-PCR, and spoligotyping. We conclude that both VNTR typing methods and FLiP typing are rapid, highly reliable, and discriminative epidemiological typing methods for M. tuberculosis and that VNTR typing is the epidemiological typing method of choice for the near future.     

Heterogeneity of structural abnormalities in the 7q31.3 approximately q34 region in myeloid malignancies

Abnormalities in the long arm of chromosome 7 are a frequent chromosomal aberration in myeloid disorders. Most studies have focused on the analysis of del(7q), demonstrating the presence of several minimal deleted regions in 7q22 approximately q31. By contrast, few studies in myeloid disorders have been devoted to the analysis of translocations, either balanced or unbalanced, involving 7q. In this study, we used fluorescence in situ hybridization (FISH) to characterize the 7q31.3 approximately q34 region (markers D7S480-D7S2227) in patients with deletion or translocation of 7q. A total of 910 cases of myeloid disorders were studied by conventional cytogenetics. Fifty-eight (6%) patients had structural aberrations of 7q. FISH studies were carried out in the 27 patients with involvement of 7q31 approximately q34: 14 cases had an acute myelogenous leukemia and 13 cases had a myelodysplastic syndrome. FISH analysis revealed the existence of high complexity in the 7q31.3 approximately q34 region in patients with unbalanced translocations. No breakpoints in 7q31.3 approximately q34 were found in the cases with deletion or balanced translocation. Nevertheless, studies of unbalanced translocations showed several breakpoints in markers D7S480-D7S2227, which delineate a commonly altered region. The complexity of 7q rearrangements suggests that a synergy of different genetic factors, rather than the alteration of a single tumor suppressor gene, could be involved in the pathogenesis of del(7q) in myeloid disorders.     

Home chaos: sociodemographic, parenting, interactional, and child correlates.

We conducted 2 studies to (a) establish the usefulness of the construct of home chaos, (b) investigate its correlates, and (c) determine the validity of the Confusion, Hubbub, and Order Scale (CHAOS) used to measure the construct in each study. Study 1 relied on a sample of European American preschoolers and their mothers and Study 2 on a sample of African American school-age children and their caregivers. Home chaos was associated with less effective parental discipline; elevated behavior problems, limited attentional focusing, and reduced ability to understand and respond to social cues in children; and reduced accuracy and efficiency in a cooperative parent-child interactional task, after controlling for potential confounds. It is concluded that (a) home chaos is not a proxy for adverse social or psychological circumstances but a useful construct in its own right; (b) home chaos is associated with multiple detrimental correlates in parents and children; and (c) the CHAOS scale provides an adequate and economical measure of home confusion and disorganization that should prove useful in clinical research with diverse populations.     

Extracellular matrix activity and caveolae events contribute to cell surface receptor activation that leads to MAP kinase activation in response to UV irradiation in cultured human keratinocytes

Activation of cell surface components has been implicated in the activation of downstream signaling cascade in response to UV irradiation, and yet the identity and the interaction of those components have been scantly documented. Accumulating evidence indicates that caveolae encapsulating caveolins is the location for those interactions. We found in cultured human keratinocytes that UV irradiation induced both caveolin-1 and EGFR phosphorylation. Filipin, a caveolae disruptive agent, inhibited UV-induced caveolin-1 activation. Na+-K+-ATPase catalyzes active transport of Na+ and K+ across plasma membrane of mammalian cells, inactivation of which has recently been shown to be involved in the activation of signal transduction pathways including MAP kinase cascade. We found in this study that UV inactivated Na+-K+-ATPase in time-dependent manner, Na+-K+-ATPase activity started to decrease 5 min post UV irradiation and reduced to 60% of its original activity within 1 h. Pretreatment with Flipin and MMP inhibitor recovered Na+-K+-ATPase activity lost by UV irradiation. ECIS analysis indicated that both EGF treatment and UV irradiation increased membrane electric activity which was inhibited by MMP inhibitor and Filipin. Further study showed that pretreatment of human keratinocytes with MMP inhibitor or Filipin inhibited UV-induced phosphorylation of p38 and JNK, which was however not observed in LnCap cells, a prostate cancer cell line lacking caveolin-1. UV irradiation also induced ectodomain shedding of HB-EGF in a time-dependent manner in keratinocytes. Collectively, we conclude that UV-induced MAP kinase activation is mediated by cell surface receptor activation due to the matrix activity and membrane caveolae function and inactivation of Na+-K+-ATPase.