Joining forces in the quest for orthologs

A report of the 24th International Conference on Yeast Genetics and Molecular Biology, Manchester, UK, 19-24 July 2009.     

An improved technique for obtaining well-spread metaphases from plants with numerous large chromosomes

Preparations that contain well-spread metaphase chromosomes are critical for plant cytogenetic analyses including chromosome counts, banding procedures, in situ hybridization, karyotyping and construction of ideograms. Chromosome spreading is difficult for plants with large and numerous chromosomes. We report here a technique for obtaining cytoplasm-free, well-spread metaphases from two Amaryllidaceae species: Sprekelia formosissima (2n = 120) and Hymenocallis howardii (2n = 96). The technique has three main steps: 1) pretreatment to cause chromosome condensation, 2) dripping onto tilted slides coated with a thin layer of pure acetic acid and 3) application of steam and acetic acid to produce cytoplasmic hydrolysis, which spreads the chromosomes.     

Brood parasitism,female condition and clutch reduction in the common eider Somateria mollisima

Conspecific brood parasitism (CBP) is an alternative reproductive tactic found in many animals with parental care. Parasitizing females lay eggs in the nests of other females (hosts) of the same species, which incubate and raise both their own and the foreign offspring. The causes and consequences of CBP are debated. Using albumen fingerprinting of eggs for accurately detecting parasitism, we here analyse its relation to female condition and clutch size in High Arctic common eiders Somateria mollissima borealis. Among 166 clutches in a Svalbard colony, 31 (19%) contained eggs from more than one female, and 40 of 670 eggs (6%) were parasitic. In 6 cases an active nest with egg(s) was taken over by another female. Many suitable nest sites were unoccupied, indicating that CBP and nest takeover are reproductive tactics, not only consequences of nest site shortage. Similarity in body mass between female categories suggests that condition does not determine whether a nesting female becomes parasitised. There was no evidence of low condition in parasites: egg size was similar in hosts and parasites, and parasitism was equally frequent early and late in the laying season. Meta‐analysis of this and 3 other eider studies shows that there is a cost of being parasitised in this precocial species: host females laid on average 7% fewer eggs than other females.     

Distribution and strain-dependent formation of volatile metabolites in the genus Ceratocystis

The accumulation of volatile metabolites in cultures of 34 strains comprising ten species of the genus Ceratocystis (Ascomycetes) has been investigated under defined culture conditions. The identified compounds include short-chain alcohols and esters, lower terpenes, terpenoids, and 2-phenylethyl acetate. Certain species can be recognized by a number of common volatiles traced in all strains, although the quantities of these constituents may vary enormously (up to a factor 1:1000) within one species. The formation of some metabolites is restricted to a few strains.     

LTBP-2 specifically interacts with the amino-terminal region of fibrillin-1 and competes with LTBP-1 for binding to this microfibrillar protein.

LTBP-2 is a matrix protein of unknown function since, unlike other LTBPs, it does not form covalent complexes with latent TGF-beta. We have previously shown that LTBP-2 has widespread association with fibrillin-containing microfibrils in developing aorta and other tissues. We have now shown that full-length human recombinant LTBP-2 specifically binds to the amino-terminal region of fibrillin-1, but not to fibrillin-2, in solid phase assays and overlay blotting. The binding was enhanced by the inclusion of 2 mM Ca2+ ions in the assay buffer and abolished by 5 mM EDTA indicating that the interaction was directly or indirectly Ca2+ ion dependent. The K(d) for the interaction was calculated from the specific binding curve as 9.4 nM. A recombinant carboxyl-terminal fragment of LTBP-2 was shown to a) bind the amino-terminal fragment of fibrillin-1 and b) block completely the binding of full length LTBP-2 to fibrillin-1. This result indicates that the major fibrillin-1 binding site resides close to the carboxyl-terminus of LTBP-2. Further competitive binding studies showed that an analogous carboxyl terminal fragment of LTBP-1 was able to block the binding of LTBP-2 to fibrillin-1 and that the C-terminal fragment of LTBP-2 could block the interaction of the LTBP-1 fragment with the fibrillin. Thus the binding site for LTBP-2 on fibrillin-1 appears to be the same or in close proximity to that for LTBP-1. Immunohistochemical analysis of developing human aorta showed distinctive but extensively overlapping distributions for LTBPs-1 and -2. Both LTBPs showed extensive co-localization with fibrillin-1 and elastic lamellae but LTBP-2 had extensive signal throughout the medial layer whereas LTBP-1 showed strong localization only in the outer medial layer. The finding indicates that there is a possibility for LTBP-2 to compete with LTBP-1 for binding to fibrillin-containing microfibrils throughout the aortic wall but particularly in the outer medial region where the LTBP-1 is predominantly located. Overall, the results support the concept that that LTBP-2 may be an indirect negative modulator for storage of the large latent TGF-beta complex on microfibrils in aorta and other fibrillin-rich tissues.     

Distinct properties of the egress‐related osmiophilic bodies in male and female gametocytes of the rodent malaria parasite Plasmodium berghei

Gametogenesis is the earliest event after uptake of malaria parasites by the mosquito vector, with a decisive impact on colonization of the mosquito midgut. This process is triggered by a drop in temperature and contact with mosquito molecules. In a few minutes, male and female gametocytes escape from the host erythrocyte by rupturing the parasitophorous vacuole and the erythrocyte membranes. Electron‐dense, oval‐shaped organelles, the osmiophilic bodies (OB), have been implicated in the egress of female gametocytes. By comparative electron microscopy and electron tomography analyses combined with immunolocalization experiments, we here define the morphological features distinctive of male secretory organelles, hereafter named MOB (male osmiophilic bodies). These organelles appear as club‐shaped, electron‐dense vesicles, smaller than female OB. We found that a drop in temperature triggers MOB clustering, independently of exposure to other stimuli. MDV1/PEG3, a protein associated with OB in Plasmodium berghei females, localizes to both non‐clustered and clustered MOB, suggesting that clustering precedes vesicle discharge. A P. berghei mutant lacking the OB‐resident female‐specific protein Pbg377 displays a dramatic reduction in size of the OB, accompanied by a delay in female gamete egress efficiency, while female gamete fertility is not affected. Immunolocalization experiments indicated that MDV1/PEG3 is still recruited to OB‐remnant structures.     

Cryo transmission X-ray imaging of the malaria parasite,P. falciparum

Cryo transmission X-ray microscopy in the “water window” of photon energies has recently been introduced as a method that exploits the natural contrast of biological samples. We have used cryo tomographic X-ray imaging of the intra-erythrocytic malaria parasite, Plasmodium falciparum, to undertake a survey of the cellular features of this important human pathogen. We examined whole hydrated cells at different stages of growth and defined some of the structures with different X-ray density, including the parasite nucleus, cytoplasm, digestive vacuole and the hemoglobin degradation product, hemozoin. As the parasite develops from an early cup-shaped morphology to a more rounded shape, puncta of hemozoin are formed; these coalesce in the mature trophozoite into a central compartment. In some trophozoite stage parasites we observed invaginations of the parasite surface and, using a selective permeabilization process, showed that these remain connected to the RBC cytoplasm. Some of these invaginations have large openings consistent with phagocytic structures and we observed independent endocytic vesicles in the parasite cytoplasm which appear to play a role in hemoglobin uptake. In schizont stage parasites staggered mitosis was observed and X-ray-dense lipid-rich structures were evident at their apical ends of the developing daughter cells. Treatment of parasites with the antimalarial drug artemisinin appears to affect parasite development and their ability to produce the hemoglobin breakdown product, hemozoin.     

The right to informed choice. A study and opinion poll of women who were or were not given the option of a sterilisation with their caesarean section

Background

In the Netherlands, caesarean sections (CSs) are rarely combined with tubal occlusion (TO), partly because discussing CS/TO near delivery is considered unethical and earlier hypothetical counselling – i.e. suppose you happen to need a CS – is rare. This results in more unintended pregnancies and is inconsistent with informed choice. We explored whether TO should indeed not be made routinely available to eligible women.

Methods and Findings

A questionnaire was mailed to 515 Para ≥2 who underwent in the past ≥1 CS. 498 (96.7%) responded. They were on average 35.3 years old, had 2.5 children, had undergone 1.6 CSs, and 3.3 years had passed since their index delivery, either a CS (393) or vaginal birth (105) after a previous CS. 87% of the 498 believed that pregnant mothers with ≥1 children should be routinely counselled about CS/TO. Indeed, 58% and 85% respectively, thought women/couples expecting their second or third child should still be given the TO option days before delivery, if omitted earlier. Counselled women, 138/498 (27.8%), were far more often satisfied than those without CS/TO option. 33/393 had a CS/TO. None indicated regret in the questionnaire. Another 119 also would have elected a CS/TO if given that option. Therefore, 152 (38.7%) of 393 Para ≥2 had or would have liked a concurrent TO. 118/119 wrote they still regretted missing this opportunity. The exception''s husband had had a vasectomy. 100/119 were good TO candidates: they were ≥28 years when they delivered an apparently healthy baby of ≥37 weeks. The current contraceptive use of these 100 suggests that this group will have at least 8 unintended pregnancies before age 50.

Conclusion

The experiences and opinions of previous potential candidates for a CS/TO do not support the reluctance of Dutch obstetricians to counsel pregnant Para ≥1 about the TO option for a (potential) CS.