The two sides of a lipid-protein story

Protein–membrane interactions play essential roles in a variety of cell functions such as signaling, membrane trafficking, and transport. Membrane-recruited cytosolic proteins that interact transiently and interfacially with lipid bilayers perform several of those functions. Experimental techniques capable of probing changes on the structural dynamics of this weak association are surprisingly limited. Among such techniques, electron spin resonance (ESR) has the enormous advantage of providing valuable local information from both membrane and protein perspectives by using intrinsic paramagnetic probes in metalloproteins or by attaching nitroxide spin labels to proteins and lipids. In this review, we discuss the power of ESR to unravel relevant structural and functional details of lipid–peripheral membrane protein interactions with special emphasis on local changes of specific regions of the protein and/or the lipids. First, we show how ESR can be used to investigate the direct interaction between a protein and a particular lipid, illustrating the case of lipid binding into a hydrophobic pocket of chlorocatechol 1,2-dioxygenase, a non-heme iron enzyme responsible for catabolism of aromatic compounds that are industrially released in the environment. In the second case, we show the effects of GPI-anchored tissue-nonspecific alkaline phosphatase, a protein that plays a crucial role in skeletal mineralization, and on the ordering and dynamics of lipid acyl chains. Then, switching to the protein perspective, we analyze the interaction with model membranes of the brain fatty acid binding protein, the major actor in the reversible binding and transport of hydrophobic ligands such as long-chain, saturated, or unsaturated fatty acids. Finally, we conclude by discussing how both lipid and protein views can be associated to address a common question regarding the molecular mechanism by which dihydroorotate dehydrogenase, an essential enzyme for the de novo synthesis of pyrimidine nucleotides, and how it fishes out membrane-embedded quinones to perform its function.     

Synthesis and properties of oligo-2'-deoxyribonucleotides containing internucleotidic phosphoramidate linkages modified with pendant groups ending with either two amino or two hydroxyl functions

Single and multiple incorporations of stereochemically pure modified dinucleoside-phosphoramidates involving substituent groups ending with bis-hydroxyethyl and bis-aminoethyl groups have been performed into pyrimidic triple helix-forming oligo-2'-deoxyribonucleotides designed to bind parallel to the purine strand of the DNA target. The ability of these modified oligo-2'-deoxyribonucleotides to form triple helices has been studied by UV-melting curve analyses, and circular dichroism. Only the oligonucleotides involving modified phosphate groups with the Rp configuration formed more stable triple helices than did the parent phosphodiester sequences. Incorporating the modifications into the third oligonucleotide strands has little effect on the structure of the triplexes. At pH 7, the incorporation of two, three or four modified phosphate groups into the third strands stabilizes the triplexes, as compared to the unmodified oligonucleotide. Stronger stabilization was observed with compounds containing linkers ending with amino functions. Stability increases with the number of modifications without being fully additive. This might be due to the different environments of the phosphate groups inside the sequence.     

Intron evolution: a statistical comparison of two models.

The two most frequently occurring explanations for the existence and distribution of introns in the genes of different species are: (1) introns are remnants of the original genetic material. (2) Introns were introduced during evolution. We construct mathematical models corresponding to these two explanations, and calculate the probabilities that the intron distribution in genes from different species coding for actin, alpha-tubulin, triosephosphate isomerase and superoxide dismutase are described by these models. In both models, the branch lengths as well as the structure of the corresponding evolutionary tree is taken into account. Every branch in the evolutionary tree is assumed to have its own individual rate of loss of introns for the first model and rate of gain of introns for the second model. These rate constants are estimated from the actual number of introns. Using the rate constants we stimulate the intron evolution and calculate the probabilities that the actual intron arrangements are produced. The results for actin and alpha-tubulin, which are the two genes we have the most data for, favor the model corresponding conjecture (1), i.e. the idea that introns are old. This contradicts the results from an earlier attempt to model intron evolution where almost the same data was used (Dibb & Newman, 1989, EMBO J. 8, 2015-2021).     

两种生境下鹅绒委陵菜无性系形态的比较

以调查统计的方法研究了矮嵩草草甸和金露梅灌丛两种生境下鹅绒委陵菜无性系的形态特性,研究结果表明：矮嵩草草甸内的鹅绒委陵菜无性系绝大多数特征参数大于金露梅灌丛内,形态可塑性较显著,这与不同生境下的资源异质性特征密切相关。研究为生境适应假说提供又一例证。     

Effects of culturing bovine oocytes either singly or in groups on development to blastocysts

In vitro maturation, fertilization and culture (IVM/IVF/IVC) of cattle oocytes from individual cows requires adapting existing culture protocols so that small numbers of oocytes can be cultured. The culture of single oocytes is desirable for correlating the relationship between follicular properties with oocyte developmental competence or for facilitating ovum pick-up procedures. In Experiment 1 we compared group and single culture under cell-free conditions on embryo development; significantly higher (P<0.001) rates of cleavage (66.4 vs 47.6%) and blastocyst formation (7.5 vs 0.5%) were observed in the group cultured oocytes. In Experiment 2 we compared group and single oocyte co-culture with granulosa cells. Although there was no effect of oocyte number on the percentage cleaving (73.1 vs 66.6%), there were significantly higher blastocyst yields (37.4 vs 10.1%) and blastocyst cell numbers (91.6 vs 66.2) in group-cultured oocytes. In Experiment 3 we examined the effect of group size (1, 5, 10, 20 and 40 oocytes) in a co-culture system using granulosa cell monolayers. The results show a difference in cleavage rates between the single cultured oocytes (66.8%) and each group of cultured oocytes, with the highest cleavage rate (81.5%) obtained in the 20-oocyte group. The blastocyst yield from both cleaved and total oocytes showed that group culture of 20 or 40 oocytes resulted in the highest number of blastocysts (32.5%), with smaller group sizes yielding significantly (P<0.05) fewer blastocysts. In Experiment 4 we examined the effects of co-culture on the development of single vs group-cultured oocytes. The results showed no significant difference (P>0.05) in the cleavage rate between single and group culture systems. No blastocysts were formed with single oocytes cultured without monolayers, while the blastocyst formation rate for those co-cultured with granulosa cells was 12.4%. Blastocyst formation was significantly higher (P < 0.006) in group co-culture on monolayers (24.2 vs 8.5%). These data indicate that oocytes cultured in groups are developmentally more competent and suggest that for optimum development oocytes need some undefined paracrine activity that is absent from the culture medium in addition to coculture with granulosa cells, which enhances development to the blastocyst stage of both group and singly cultured oocytes.     

Cross pathogenicity studies with isolates of Fusarium oxysporum from either cucumber or watermelon pathogenic to both crop species*

Isolates of Fusarium oxysporum from Abaco, the Bahamas, whether obtained from wilted plants of cucumber (Cucumis sativus) or watermelon (Citrullus lanatus), were pathogenic to cucumber, watermelon, and cantaloupe (Cucumis melo var. reticulatus). The West Indian gherkin (Cucumis anguria), pumpkin (Cucurbita pepo), and three cultivars of summer squash (C. pepo var. melopepo) were not susceptible. Strains of F. oxysporum from wilted cucumber or watermelon plants from Florida were highly pathogenic only to their original host species and are regarded as different formae speciales.     

The erythrocyte insulin receptor response to either feeding or a short fast

We have examined insulin binding to the erythrocyte insulin receptor in normal males following a short fast and a test meal or glucose load. The binding increased following the fast. This increase appeared to be due to increased receptor affinity. Following the meal or glucose load no change in insulin binding was detected. These results are compared with conflicting data in the literature and possible technical explanations for the discrepancies are explored.     

Spermatozoan morphology as a character for tardigrade systematics: comparison with sclerified parts of animals and eggs in eutardigrades

The male gamete, a cell widely used for evaluating phylogeny in different animal groups, remains relatively unknown in tardigrades. In this paper the spermatozoa of thirteen species of eutardigrades among four genera and three families is evaluated in order to determine whether sperm morphology can be used as a taxonomic character. Spermatozoa of Amphibolus volubilis and A. weglarskae are very similar and this resemblance is congruent with the remarkable similarity of sclerified parts of the species. In addition, the spermatozoa of eight species of the genus Macrobiotus were examined yielding two groups showing strong intragroup similarities. The first group includes M. pseudohufelandi, M. sandrae, M. macrocalix, M. terminals and M.joannae , and the second M. richtersi, M. areolatus and M. harmsworthi. Again, these groupings were congruent with those determined by analysis of sclerified structures. In contrast, a marked similarity was found between the spermatozoa ot Diphascon (Adropion) scoticum and Platicrista angustata , whereas Diphascon (Diphascon) humicus was appreciably different from both species. Resemblances found in this study between spermatozoa and sclerified body parts suggests that the spermatozoa are suitable characters for use in systematic studies.     

Nup98 localizes to both nuclear and cytoplasmic sides of the nuclear pore and binds to two distinct nucleoporin subcomplexes

The vertebrate nuclear pore is an enormous structure that spans the double membrane of the nuclear envelope. In yeast, most nucleoporins are found symmetrically on both the nuclear and cytoplasmic sides of the structure. However, in vertebrates most nucleoporins have been localized exclusively to one side of the nuclear pore. Herein, we show, by immunofluorescence and immunoelectron microscopy, that Nup98 is found on both sides of the pore complex. Additionally, we find that the pore-targeting domain of Nup98 interacts directly with the cytoplasmic nucleoporin Nup88, a component of the Nup214, Nup88, Nup62 subcomplex. Nup98 was previously described to interact with the nuclear-oriented Nup160, 133, 107, 96 complex through direct binding to Nup96. Interestingly, the same site within Nup98 is involved in binding to both Nup88 and Nup96. Autoproteolytic cleavage of the Nup98 C terminus is required for both of these binding interactions. When cleavage is blocked by a point mutation, a minimal eight amino acids downstream of the cleavage site is sufficient to prevent most binding to either Nup96 or Nup88. Thus, Nup98 interacts with both faces of the nuclear pore, a localization in keeping with its previously described nucleocytoplasmic shuttling activity.     

Identification of a polyphosphoinositide-modulated domain in gelsolin which binds to the sides of actin filaments

Gelsolin is a Ca2+- and polyphosphoinositide-modulated actin-binding protein which severs actin filaments, nucleates actin assembly, and caps the "barbed" end of actin filaments. Proteolytic cleavage analysis of human plasma gelsolin has shown that the NH2-terminal half of the molecule severs actin filaments almost as effectively as native gelsolin in a Ca2+-insensitive but polyphosphoinositide-inhibited manner. Further proteolysis of the NH2-terminal half generates two unique fragments (CT14N and CT28N), which have minimal severing activity. Under physiological salt conditions, CT14N binds monomeric actin coupled to Sepharose but CT28N does not. In this paper, we show that CT28N binds stoichiometrically and with high affinity to actin subunits in filaments, suggesting that it preferentially recognizes the conformation of polymerized actin. Analysis of the binding data shows that actin filaments have one class of CT28N binding sites with Kd = 2.0 X 10(-7) M, which saturates at a CT28N/actin subunit ratio of 0.8. Binding of CT28N to actin filaments is inhibited by phosphatidylinositol 4,5-bisphosphate micelles. In contrast, neither CT14N nor another actin-binding domain located in the COOH-terminal half of gelsolin form stable stoichiometric complexes with actin along the filaments, and their binding to actin monomers is not inhibited by PIP2. Based on these observations, we propose that CT28N is the polyphosphoinositide-regulated actin-binding domain which allows gelsolin to bind to actin subunits within a filament before serving.     

A comparison of management strategies for conservation with regard to population fitness

Computer simulations have been carried out tocompare, under realistic genetic models, twomethods proposed in the literature to retaingenetic diversity in conservation programmes.In a two-step method, contributions of parentsare set up to produce minimum coancestry(kinship) among the offspring, and this isindependent of the mating system subsequentlyapplied. In a single-step method,contributions and matings are decidedsimultaneously in order to minimise coancestry.The comparison is made in terms of maintainedgenetic diversity and in terms of populationfitness. We conclude that the two methodsmaintain approximately the same geneticdiversity but the latter induces higher levelsof inbreeding, reducing the fitness of thepopulation. Avoidance of close relatives'matings improves this latter method, but thefitness levels do not reach those of thetwo-step scheme. We also investigate theperformances of different mating strategies incombination with minimum coancestry (two-stepmethod), concluding that these mating systemsdo not substantially affect the effectivenessof the management. Finally, we illustrate howminimum group coancestry can be restrictedto a minimum loss of fitness, if a measure ofthis is available for the individuals.