Molecular mechanisms of determination in Drosophila

A number of Drosophila proteins have been identified that play key roles in the establishment of active or inactive states of selector gene expression. Interactions between these proteins and their target selector genes are beginning to be understood, shaping our molecular view as to how stable determination of cells is achieved.     

Unusual sialilation of three different rare genetic variants of serum DBP: Gc 1A17, Gc 1A16, and Gc 1A11

Summary The proteins of three anodal Gc1 variants, Gc 1A16, 1A11, and 1A17, are characterized by the most acidic isoelectric points observed so far among the different Gc mutants. Stepwise removal of N-acetylneuraminic acid (NANA) by treatment with neuraminidase was performed to estimate the degree of sialilation of these Gc variants. The results indicate that both proteins, the anodal and the cathodal component of these Gc 1 mutants, carry sialic acid residues. This observation is remarkable in so far as usually only the anodal component of the Gc 1 protein contains NANA and only a single residue. From the experiments carried out it can be deduced that Gc 1A16 has two NANA residues in the anodal and one NANA residue in the cathodal component. Gc 1A16 was found in four members of three generations in a Danish family; the variant segregated as a Mendelian trait. More difficult to interprete are the results obtained with the variants Gc 1A11 and Gc 1A17. Gc 1A11 probably has three NANA residues in the anodal and two NANA residues in the cathodal component. Gc 1A11 has been observed in two mother-child pairs and is presumably also a simple genetic trait. Gc 1A17 has also several NANA residues in both Gc proteins; it is suggested that the anodal component has either three or four NANA residues and the cathodal component either two or three NANA residues. Family information on this variant is not yet available.     

Higher-Order Looping and Nuclear Organization of Tcra Facilitate Targeted RAG Cleavage and Regulated Rearrangement in Recombination Centers

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Highlights? RAG-dependent monoallelic loop formation is linked to monoallelic RAG cleavage ? RAG enrichment, cleavage, and higher-order loop formation occur at the 3′ end of Tcra ? Looping out is a determinant of directed RAG targeting ? ATM-mediated control of looping out is linked to the maintenance of genome stability     

Modelled population growth based on reproduction differs from life tables based on age determination in Danish raccoon dogs ( Nyctereutes procyonoides )

The population of raccoon dogs (Nyctereutes procyonoides) in Denmark has increased rapidly from 1995 when the first was recorded until today where 3291 raccoon dogs are trapped, shot by hunters or road killed. The aims of this study are to present the first data on reproduction and life tables of raccoon dogs in Denmark and to compare mortality from modelled life tables with game bag records and sampled raccoon dogs in different age groups. In this study, the uteri of 89 adult females (> 10 months) were examined for placental scars (PSC), and 561 individuals (289 males, 272 females) were aged using pulp cavity width and dental lines in canine teeth. The litter size of raccoon dogs in Denmark is to date the largest litter size recorded in the wild (mean ± SE) 10.8 ± 0.4, range 1–16 pubs and fecundity 8.4 ± 0.6 pubs. The percent-reproducing females are 78–83%, based on dark and all PSC, respectively. A significant difference was found between the proportion of individuals composing the different age groups based on age determination of individuals collected (Ntage) and the modelled number of individuals in age groups based on fecundity and different mortality rate (Ntmodel), X2 = 8, p < 0.05. The discrepancy between the relatively high reproduction and lifetables may be due to older and more experienced animals that avoid culling. A low population density in a newly founded Danish population of raccoon dogs, together with a milder climate where raccoon dogs can forage during the winter, may cause an exceptionally high reproduction in Danish raccoon dogs.     

Sexual dimorphism in rat left atrial function and response to adrenergic stimulation

A number of investigations in humans and animals suggest that there may be intrinsic sex-associated differences in cardiac function. Using left atrial preparations from male and female rat hearts, we examined differences in myocardial function and response to adrenergic agonists. Contractile parameters were measured in isolated atria by conventional isometric methods in the absence or presence of isoproterenol or phenylephrine. Responsiveness to Ca²⁺ was measured in detergent-skinned atrial fibers and actomyosin ATPase activity was measured in isolated myofibrils. Tetanic contractions were generated by treating the atrium with ryanodine followed by high frequency stimulation. Developed force was greater and maximal rates of contraction and relaxation were more rapid in the female atrium. The relationship between Ca²⁺ concentration and force in both intact atria and detergent-skinned atrial fibers in females fell to the left of that for males. At low Ca²⁺ concentrations, skinned fibers from female atria generated more force and myofibrils from female atria had higher myosin ATPase activity than males. Tetanic contraction in the presence of high extracellular Ca²⁺ was greater in female atria. Male atrium had larger inotropic responses to isoproterenol and to phenylephrine, but drug-elicited cAMP and inositol phosphate production did not differ between sexes. The results demonstrate sex-related differences in atrial function that can be partially explained by greater myofibrillar Ca²⁺-sensitivity in females. A potential contribution of sarcolemmal Ca2+ influx is suggested by greater tetanic contraction in ryanodine-treated female atrium. The larger response of males to adrenergic stimulation does not appear to be explained by higher production of relevant second messengers. Future studies will investigate the role of sex hormones in these sexually dimorphic responses and may indicate a need for gender-specific therapeutic interventions for myocardial dysfunction.     

Condensation of interphase chromatin in nuclei of synchronized chinese hamster ovary (CHO-K1) cells

Reversibly permeabilized cells have been used to visualize interphase chromatin structures in the presence and absence of biotinylated nucleotides. By reversing permeabilization, it was possible to confirm the existence of a flexible chromatin folding pattern through a series of transient geometric forms such as supercoiled, circular forms, chromatin bodies, thin and thick fibers, and elongated chromosomes. Our results show that the incorporation of biotin-11-dUTP interferes with chromatin condensation, leading to the accumulation of decondensed chromatin structures. Chromatin condensation without nucleotide incorporation was also studied in cell populations synchronized by centrifugal elutriation. After reversal of permeabilization, nuclei were isolated and chromatin structures were visualized after DAPI staining by fluorescent microscopy. Decondensed veil-like structures were observed in the early S phase (at an average C-value of 2.21), supercoiled chromatin later in the early S (2, 55 C), fibrous structures in the early mid S phase (2, 76 C), ribboned structures in the mid-S phase (2, 98 C), continuous chromatin strings later in the mid-S phase (3,28), elongated prechromosomes in the late S-phase (3, 72 C), precondensed chromosomes at the end and after the S phase (3, 99 C). Fluorescent microscopy revealed that neither interphase nor metaphase chromosomes are separate entities but form a linear array arranged in a semicircle. Linear arrangement was confirmed by computer image analysis.     

Loss of Drosophila borealin causes polyploidy, delayed apoptosis and abnormal tissue development

The chromosomal passenger complex (CPC) is a key regulator of mitosis in many organisms, including yeast and mammals. Its components co-localise at the equator of the mitotic spindle and function interdependently to control multiple mitotic events such as assembly and stability of bipolar spindles, and faithful chromosome segregation into daughter cells. Here, we report the first detailed characterisation of a CPC mutation in Drosophila, using a loss-of-function allele of borealin (borr). Like its mammalian counterpart, Borr colocalises with the CPC components Aurora B kinase and Incenp in mitotic Drosophila cells, and is required for their localisation to the mitotic spindle. borr mutant cells show multiple mitotic defects that are consistent with loss of CPC function. These include a drastic reduction of histone H3 phosphorylation at serine 10 (a target of Aurora B kinase), a pronounced attenuation at prometaphase and multipolar spindles. Our evidence suggests that borr mutant cells undergo multiple consecutive abnormal mitoses, producing large cells with giant nuclei and high ploidy that eventually apoptose. The delayed apoptosis of borr mutant cells in the developing wing disc appears to cause non-autonomous repair responses in the neighbouring wild-type epithelium that involve Wingless signalling, which ultimately perturb the tissue architecture of adult flies. Unexpectedly, during late larval development, cells survive loss of borr and develop giant bristles that may reflect their high degree of ploidy.     

Cardiovascular effects of epinephrine during rewarming from hypothermia in an intact animal model.

Rewarming from accidental hypothermia is often complicated by "rewarming shock," characterized by low cardiac output (CO) and a sudden fall in peripheral arterial pressure. In this study, we tested whether epinephrine (Epi) is able to prevent rewarming shock when given intravenously during rewarming from experimental hypothermia in doses tested to elevate CO and induce vasodilation, or lack of vasodilation, during normothermia. A rat model designed for circulatory studies during experimental hypothermia and rewarming was used. A total of six groups of animals were used: normothermic groups 1, 2, and 3 for dose-finding studies, and hypothermic groups 4, 5, and 6. At 20 and 24 degrees C during rewarming, group 4 (low-dose Epi) and group 5 (high-dose Epi) received bolus injections of 0.1 and 1.0 microg Epi, respectively. At 28 degrees C, Epi infusion was started in groups 4 and 5 with 0.125 and 1.25 microg/min, respectively. Group 6 served as saline control. After rewarming, both CO and stroke volume were restored in group 4, in contrast to groups 5 and 6, in which both CO and stroke volume remained significantly reduced (30%). Total peripheral resistance was significantly higher in group 5 during rewarming from 24 to 34 degrees C, compared with groups 4 and 6. This study shows that, in contrast to normothermic conditions, Epi infused during hypothermia induces vasoconstriction rather than vasodilation combined with lack of CO elevation. The apparent dissociation between myocardial and vascular responses to Epi at low temperatures may be related to hypothermia-induced myocardial failure and changes in temperature-dependent adrenoreceptor affinity.     

Zinc fluxes into developing barley grains: use of stable Zn isotopes to separate root uptake from remobilization in plants with contrasting Zn status

Background and Aims

Zn imported into developing cereal grains originates from either de novo Zn uptake by the roots or remobilization of Zn from vegetative tissues. The present study was focused on revealing the quantitative importance of the two pathways for grain Zn loading and how their relative contribution varies with the overall plant Zn status.

Methods

The stable isotope ⁶⁷Zn was used to trace Zn uptake and remobilization fluxes in barley (Hordeum vulgare L.) plants growing in hydroponics at 0.1?μM (low Zn), 1.5?μM (medium Zn) or 5?μM Zn (high Zn). When grain development reached 15?days after pollination the Zn source was changed to an enriched ⁶⁷Zn isotope and plants were harvested after 6 to 48?h. Zn concentrations and isotope ratios were determined using Inductively Coupled Plasma-Mass Spectrometry (ICP-MS).

Results

Plants with low Zn status absorbed 3-fold more Zn than plants with medium or high Zn status when roots were exposed to an external concentration of 1.5?μM ⁶⁷Zn. Stems and ears were the primary recipients of the de novo incorporated Zn with preferential allocation to the developing grains over time. The leaves received in all cases a very small proportion (<5?%) of the newly absorbed Zn and the proportion did not increase over time. Zn fluxes derived from uptake and remobilization were almost equal in plants with low Zn status, while at high Zn status remobilization delivered 4 times more Zn to the developing grains than did root Zn uptake.

Conclusions

Stable isotopes in combination with ICP-MS provided a strong tool for quantification of Zn fluxes in intact plants. The importance of Zn remobilization compared to de novo root absorption of Zn increased with increasing plant Zn status. Very little de novo absorbed Zn was translocated to the leaves during generative growth stages.