Toward a high resolution 2-DE profile of the normal human liver proteome using ultra-zoom gels

The human liver is the largest organ in the body and has many important physiological functions. A global analysis of human liver proteins is essential for a better understanding of the molecular basis of the normal functions of the liver and of its diseases. As part of the Human Liver Proteome Project (HLPP), the goal of the present study was to visualize and detect as many proteins as possible in normal human livers using two-dimensional gel electrophoresis (2-DE). We have constructed a reference map of the proteins of human normal liver that can be used for the comprehensive analysis of the human liver proteome and other related research. To improve the resolution and enhance the detection of low abundance proteins, we developed and optimized narrow pH range ultra-zoom 2-DE gels. High resolution patterns of human liver in pH gradients 4.5–5.5, 5–6, 5.5–6.7, 6–9 and 6–11 are presented. To improve the poor resolution in the alkaline pH range of 2-DE gels, we optimized the isoelectric focusing protocol by including sample application using cup loading at the anode and incorporating 1.2% hydroxyethyl disulfide, 15% 2-propanol and 5% glycerol in the rehydration buffer. Using the optimized protocol, we obtained reproducibly better resolution in both analytical and preparative 2-DE gels. Compared with the 2386 and 1878 protein spots resolved in the wide range 3–10 and 4–7 pH gradients respectively, we obtained 5481 protein spots from the multiple (overlapping) narrow pH range ultra-zoom gels in the range of pH 4.5–9. The visualized reference map of normal human liver proteins presented in this paper will be valuable for comparative proteomic research of the liver proteome.     

The distribution–abundance (density) relationship: its form and causes in a tropical mammal order, Primates

Aim Across a wide variety of organisms, taxa with high local densities (abundance) have large geographical ranges (distributions). We use primatology's detailed knowledge of its taxon to investigate the form and causes of the relationship in, unusually for macroecological analysis, a tropical taxon. Location Africa, Central and South America, Asia, Madagascar. Methods To investigate the form of the density–range relationship, we regressed local density on geographical range size, and also on female body mass, because in the Primates, density correlates strongly with mass. To investigate the biological causes of the relationship, we related (1) abundance (density × range size) and (2) residuals from the density–range regression lines to various measures of (i) resource use, (ii) reproductive rate and (iii) potential specialization. All data are from the literature. Analyses were done at the level of species (n = 140), genera (n = 60) and families/subfamilies (n = 17). We present various levels of results, including for all data, after omission of outlier data, after correction for phylogenetic dependence, and after Bonferroni correction of probabilities for multiple comparisons. Results Regarding the form of the relationship, Madagascar primates are clear outliers (high densities in small ranges). Among the remaining three realms, the relation of density to range is weak or non‐existent at the level of species and genera. However, it is strong, tight and linear at the level of families/subfamilies (r² = 0.6, F_1,10 = 19, P < 0.01). Although among primates, density is very significantly related to mass, at no taxonomic level is range size related to body mass. Consequently, removing the effects of mass makes little to no difference to density–range results. Regarding the biology of the relationship, only traits indicative of specialization are associated with abundance (meaning numbers): rare taxa are more specialized than are abundant taxa. The association is largely via range size, not density. Across families, no traits correlate significantly with the density–range relationship, nor with deviations from it, despite the strength of the relationship at this taxonomic level. Main conclusions We suggest that in macroecology, analysis at taxonomic levels deeper than that of the relatively ephemeral species can be appropriate. We argue that the several purely methodological explanations for the positive density–range size relationship in primates can be rejected. Of the various biological hypotheses, those having to do with specialization–generalization seem the only applicable ones. The fact that the relationship is entirely via range size, not via density, means that while we might have a biology of range size, we do not yet have one of the density–geographical range relationship. It is probably time to search for multivariate explanations, rather than univariate ones. However, we can for the first time, for at least primates, suggest that any association of abundance or range size with specialization is via the number of different subtaxa, not the average degree of specialization of each subtaxon. The implication for conservation is obvious.     

FIRST EVIDENCE OF PALYTOXIN ANALOGUES FROM AN OSTREOPSIS MASCARENENSIS (DINOPHYCEAE) BENTHIC BLOOM IN SOUTHWESTERN INDIAN OCEAN1

Benthic dinoflagellates of the genus Ostreopsis Schmidt are common in tropical and subtropical water, and some species produce toxins potentially involved in human intoxication events. A benthic bloom of Ostreopsis mascarenensis Quod was observed near Rodrigues Island during a survey of benthic dinoflagellates in the southwestern Indian Ocean. The morphology of O. mascarenensis was studied by LM and SEM. Preliminary screening of a crude extract of an O. mascarenensis bloom revealed neurotoxicity in mice similar to that induced by palytoxin. After partition of the crude extract, the highest toxicity was retained in the butanol‐soluble fraction, which retained hemolytic activity suggestive of palytoxin analogues. Two new toxins, mascarenotoxin‐A and ‐B, were resolved from this fraction by HPLC coupled to a diode array detector. The closed mass spectrum profile and fragmentation pattern obtained by advanced nano–electrospray ionization quadrupole time‐of‐flight mass spectrometry between purified toxins and a reference palytoxin confirmed the mascarenotoxins as palytoxin analogues. These results were confirmed by tandem mass spectrometry with the identification of specific fragment ion m/z 327. An on‐line liquid chromatography protocol coupled to tandem mass spectrometry was developed for detection of these palytoxin analogues. The present study describes the first purification, chemical, and toxicological characterization of new palytoxin analogues isolated from a benthic bloom of O. mascarenensis. These results suggest that O. mascarenensis, which is largely distributed in the southwestern Indian Ocean, could be a source of palytoxin poisoning in this tropical area.     

Indigenous Studies in the Elementary Curriculum: A Cautionary Hawaiian Example

This article uses a Native Hawaiian example to raise difficult questions about the role and responsibility of non-Indigenous educators in teaching and supporting Indigenous studies. It challenges educators and educational researchers to think closely about how they might serve as allies in Indigenous struggles for self-determination.     

Comparative fecundity and survival rates of Phlebotomus papatasi sandflies membrane fed on blood from eight mammal species

Female sandflies, Phlebotomus papatasi (Scopoli) (Diptera: Psychodidae), were fed via chicken membrane on heparinized blood from eight species of mammal (human, horse, cow, pig, dog, rabbit, guinea-pig, hamster) and their reproductive success rates were compared. No appreciable differences between those fed on human and animal blood were detected with respect to the proportion of flies that fed successfully, mortality-rate within 24h, number of eggs laid per blood-fed female or egg viability. When mass-rearing sandflies for research purposes, membrane-feeding avoids practical difficulties encountered if sandflies are allowed to feed on live hosts (i.e. anaesthesia, distress from handling and postfeeding inflammation) and reduction of sandfly fecundity due to host antibody interference. Use of animal blood also eliminates risks of accidental transmission of human blood-borne pathogens, e.g. hepatitis B and human immunodeficiency virus (HIV), and is less expensive than maintenance of animals and their preparation for sandfly feeding.     

Snapshots of the RNA editing machine in trypanosomes captured at different assembly stages in vivo

Mitochondrial pre‐messenger RNAs in kinetoplastid protozoa are substrates of uridylate‐specific RNA editing. RNA editing converts non‐functional pre‐mRNAs into translatable molecules and can generate protein diversity by alternative editing. Although several editing complexes have been described, their structure and relationship is unknown. Here, we report the isolation of functionally active RNA editing complexes by a multistep purification procedure. We show that the endogenous isolates contain two subpopulations of ～20S and ～35–40S and present the three‐dimensional structures of both complexes by electron microscopy. The ～35–40S complexes consist of a platform density packed against a semispherical element. The ～20S complexes are composed of two subdomains connected by an interface. The two particles are structurally related, and we show that RNA binding is a main determinant for the interconversion of the two complexes. The ～20S editosomes contain an RNA‐binding site, which binds gRNA, pre‐mRNA and gRNA/pre‐mRNA hybrid molecules with nanomolar affinity. Variability analysis indicates that subsets of complexes lack or possess additional domains, suggesting binding sites for components. Together, a picture of the RNA editing machinery is provided.     

Effect of ectomycorrhizal infection on growth and photosynthetic characteristics of <Emphasis Type="Italic">Pinus densiflora</Emphasis> seedlings grown under elevated CO<Subscript>2</Subscript> concentrations

The effect of ectomycorrhizal Pisolithus tinctorius (Pt) infection was studied on the growth and photosynthetic characteristics of Pinus densiflora seedlings grown at ambient (360 µmol mol⁻¹, AC) and elevated (720 µmol mol⁻¹, EC) CO₂ concentrations. After 18 weeks, Pt inoculation had led to significantly increased dry mass and stem diameter of P. densiflora at both CO₂ concentrations, relative to non-inoculated seedlings. Moreover, EC significantly increased the ectomycorrhizal development. The phosphate content in needles inoculated with Pt was about three times higher than without inoculation at both CO₂ concentrations. The PAR saturated net photosynthetic rates (P _sat) of P. densiflora inoculated with Pt were clearly higher than for control seedlings at both CO₂ concentrations, and the maximum net photosynthetic rate (P _N) at saturated CO₂ concentration (P _max) was higher than in controls. Moreover, the carboxylation efficiency (CE) and RuBP regeneration rate of the P _N/C _i curve for P. densiflora inoculated with Pt were significantly higher than for non-inoculated seedlings at both CO₂ concentrations, especially at EC. The water use efficiency (WUE) of seedlings inoculated with Pt grown at EC was significantly raised. Allocation of photosynthates to roots was greater in Pt inoculated pine seedlings, because of the enhanced activity of ectomycorrhiza associated with seedlings at EC. Moreover, P _N of non-inoculated seedlings grown for 18 weeks at EC tended to be down regulated; in contrast, Pt inoculated seedlings showed no down-regulation at EC. The activity of ectomycorrhiza may therefore be enhanced physiological function related to water and phosphate absorption in P. densiflora seedlings at EC.This study was partly sponsored by the Ministry of Education, Sport, Culture, Science and Technology of Japan (RR2002, Basic Research B and Sprout study).     

Leaf development and photosynthetic properties of three tropical tree species with delayed greening

Leaf developmental patterns were characterized for three tropical tree species with delayed greening. Changes in the pigment contents, photosynthetic capacity, stomata development, photosystem 2 efficiency, rate of energy dissipation, and the activity of partial protective enzymes were followed in developing leaves in an attempt to elucidate the relative importance of various photoprotective mechanisms during leaf ontogeny. Big leaves of Anthocephalus chinensis, a fast-growing light demanding species, expanded following an exponential pattern, while relatively small leaves of two shade-tolerant species Litsea pierrei and Litsea dilleniifolia followed a sigmoidal pattern. The juvenile leaves of A. chinensis and L. pierrei contained anthocyanin located below the upper epidermis, while L. dilleniifolia did not contain anthocyanin. Leaves of A. chinensis required about 12 d for full leaf expansion (FLE) and photosynthetic development was delayed 4 d, while L. pierrei and L. dilleniifolia required 18 or 25 d for FLE and photosynthetic development was delayed 10 or 15 d, respectively. During the leaf development the increase in maximum net photosynthetic rate was significantly related to changes in stomatal conductance and the leaf maturation period was positively related to the steady-state leaf dry mass per area for the three studied species. Dark respiration rate of leaves at developing stages was greater, and pre-dawn initial photochemical efficiency was lower than that of mature leaves. Young leaves displayed greater energy dissipation than mature leaves, but nevertheless, the diurnal photoinhibition of young L. dilleniifolia leaves was higher than that of mature leaves. The young red leaves of A. chinensis and L. pierrei with high anthocyanin contents and similar diurnal photoinhibition contained more protective enzymes (superoxide dismutase, ascorbate peroxidase) than mature leaves. Consequently, red leaves may have higher antioxidant ability.     

Functional morphology of the cephalopod buccal mass: a novel joint type

The arrangement of the musculature and connective tissues of the buccal mass of the coleoid cephalopods Octopus bimaculoides, Sepia officinalis, and Loliguncula brevis was examined using dissection and histology. Serial sections in three mutually perpendicular planes were used to identify the muscles and connective tissues responsible for beak movements and stability and to describe their morphology and fiber trajectories. Four major beak muscles were identified: the anterior, posterior, superior, and lateral mandibular muscles. The anterior, posterior, and superior mandibular muscles connect the upper beak and the lower beak. Although the lateral mandibular muscles originate on the upper beak, they do not connect to the lower beak and instead insert on a connective tissue sheath surrounding the buccal mass. Examination of the fibers of the lateral mandibular muscles reveals that they have the organization of a muscular hydrostat, with muscle fibers oriented in three mutually perpendicular orientations. Although the beaks are capable of complex opening, closing, and shearing movements, they do not contact one another and are instead connected only by the musculature of the buccal mass. Based on the morphological analysis and observations of freshly dissected beaks undergoing the stereotyped bite cycle, the functional role of the beak muscles is hypothesized. The anterior and superior mandibular muscles are likely responsible for beak closing and shearing movements. The posterior mandibular muscle is likely also involved in beak closing, but may act synergistically with the lateral mandibular muscles to open the beaks. The lateral mandibular muscles may use a muscular-hydrostatic mechanism to control the location of the pivot between the beaks and to generate the force required for beak opening. The lack of contact between the beaks and the morphology of the lateral mandibular muscles suggests that the buccal mass of coleoid cephalopods may represent a previously unexamined flexible joint mechanism. The term "muscle articulation" is proposed here to denote the importance of the musculature in the function of such a joint.