Total Polysaccharide Content of Developing Pollen in Two Angiosperm Species

The PAS reaction was used to stain total insoluble polysaccharides in whole pollen grains of anthers of Borago officinalis and Lycopersicum peruvianum from early microspore to ripe pollen stage. Total polysaccharide content was quantified with a microdensitometer: in Borago officinalis values showed two peaks, one during the microspore and the other during the binucleate stage; in Lycopersicum, there was a single peak during the latter stage. In both species total polysaccharide content decreased markedly in the last stage of pollen development before anthesis, when starch was hydrolysed and the polysaccharide reserves of the pollen transferred to the cytoplasm. Pollen grain volume was also determined at the various stages. It was found to increase, though with a different pattern in the two species, and to decrease before dehiscence. The results are discussed in terms of cytoembryological data of pollen grain development.     

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

The investigation of the intracellular protein levels of bacterial species is of importance to understanding the pathogenic mechanisms of diseases caused by these organisms. Here we describe a procedure for protein extraction from Burkholderia species based on mechanical lysis using glass beads in the presence of ethylenediamine tetraacetic acid and phenylmethylsulfonyl fluoride in phosphate buffered saline. This method can be used for different Burkholderia species, for different growth conditions, and it is likely suitable for the use in proteomic studies of other bacteria. Following protein extraction, a two-dimensional (2-D) gel electrophoresis proteomic technique is described to study global changes in the proteomes of these organisms. This method consists of the separation of proteins according to their isoelectric point by isoelectric focusing in the first dimension, followed by separation on the basis of molecular weight by acrylamide gel electrophoresis in the second dimension. Visualization of separated proteins is carried out by silver staining.     

Identification of Two Distinct Populations of Protein Kinase C in Rat Brain Membranes

The regulatory enzyme protein kinase C (PKC) is proposed to be activated on its translocation from the cytosol to the membrane. However, a portion of the native activity is always associated with the membrane fraction. Using a noninvasive procedure to extract this endogenous activity from rat brain membranes, it has been possible to characterize the activity in a partially purified reconstituted system bearing resemblance to the in vivo system. Two subpopulations of membrane-associated PKC were identified and characterized at the level of activation, inhibition, and isozyme immunologic characteristics and chromatographic properties. One peak had properties similar to those of cytosolic PKC, whereas the second population, extracted as protein-lipid complexes, had considerable constitutive activity that could be stimulated further on addition of PKC activators. This latter activity was relatively resistant to staurosporine inhibition and phorbol ester treatment, but it phosphorylated the exogenous PKC substrates, histone 1 and the epidermal growth factor receptor peptide KTRLRR. The constitutive activity was totally dependent on its endogenous associated lipids coextracted by the solubilization procedure. The ratio between these two populations was ontogenetically regulated and modulated by phorbol ester treatment, suggesting that different PKC populations may serve unique functions in the rat brain regulated by the lipid environment. Analyses of the phospholipids extracted in these protein-lipid complexes showed differences in the major classes correlating to age. However, apart from a markedly lower cholesterol content in these complexes, no direct relationship between a specific lipid composition and the amount of constitutive PKC activity was evident.     

Protein Profiling in Hepatocellular Carcinoma by Label-Free Quantitative Proteomics in Two West African Populations

Background

Hepatocellular Carcinoma is the third most common cause of cancer related death worldwide, often diagnosed by measuring serum AFP; a poor performance stand-alone biomarker. With the aim of improving on this, our study focuses on plasma proteins identified by Mass Spectrometry in order to investigate and validate differences seen in the respective proteomes of controls and subjects with LC and HCC.

Methods

Mass Spectrometry analysis using liquid chromatography electro spray ionization quadrupole time-of-flight was conducted on 339 subjects using a pooled expression profiling approach. ELISA assays were performed on four significantly differentially expressed proteins to validate their expression profiles in subjects from the Gambia and a pilot group from Nigeria. Results from this were collated for statistical multiplexing using logistic regression analysis.

Results

Twenty-six proteins were identified as differentially expressed between the three subject groups. Direct measurements of four; hemopexin, alpha-1-antitrypsin, apolipoprotein A1 and complement component 3 confirmed their change in abundance in LC and HCC versus control patients. These trends were independently replicated in the pilot validation subjects from Nigeria. The statistical multiplexing of these proteins demonstrated performance comparable to or greater than ALT in identifying liver cirrhosis or carcinogenesis. This exercise also proposed preliminary cut offs with achievable sensitivity, specificity and AUC statistics greater than reported AFP averages.

Conclusions

The validated changes of expression in these proteins have the potential for development into high-performance tests usable in the diagnosis and or monitoring of HCC and LC patients. The identification of sustained expression trends strengthens the suggestion of these four proteins as worthy candidates for further investigation in the context of liver disease. The statistical combinations also provide a novel inroad of analyses able to propose definitive cut-offs and combinations for evaluation of performance.     

Changes in Whistle Structure of Two Dolphin Species During Interspecific Associations

Dolphin communicative signals show great plasticity. Dolphins modify signal structure to cope with their environment, in response to stress, and in some species to mimic group members. Hence, whistle structure variations may offer insights to interspecific associations among dolphin species, which although temporal and opportunistic are common. In this study, I test the hypothesis that interspecific interactions influence dolphin whistle structure, particularly during social events. The study took place in the Southern Caribbean coast of Costa Rica, where interspecific associations of the distantly related Guyana and Bottlenose dolphins occur on daily basis. The results indicate that interspecific groups emit whistles that show intermediate whistle structure compared to whistles emitted in intraspecific groups. This pattern is seen during social interactions between species, but not when interspecific groups are traveling. Social events in interspecific groups were of antagonistic nature, where Bottlenose dolphins isolated and harassed one or two Guyana dolphins. Contour data suggest that the most vocal species during these encounters was the Guyana dolphin. Therefore, the observed modifications in whistles structure likely reflect a stress response by the Guyana dolphins. Another alternative explanation includes signal convergence between interacting species. However, to understand the nature of these potential modifications, future studies should combine acoustic tags and directional recording systems to follow the vocalizing animals. Despite the shortcomings of this study, it provides some of the first insights into dolphin interspecific communication, providing evidence of overall signal change during interspecific interactions.     

Parallel Patterns of Morphological and Behavioral Variation among Host-Associated Populations of Two Gall Wasp Species

A powerful approach to address the general factors contributing to ecological speciation is to compare distantly related taxa that inhabit the same selective environments. In this design, similarities among taxa can elucidate general mechanisms of the process whereas differences may uncover specific factors important to the process for individual taxa. Herein, we present evidence of parallel patterns of morphological and behavioral variation among host-associated populations of two species of cynipid gall wasps, Belonocnema treatae and Disholcaspis quercusvirens, that each exhibit a life cycle intimately tied to the same two host plant environments, Quercus geminata and Q. virginiana. Across both gall-former species we find consistent differences in body size and gall morphology associated with host plant use, as well as strong differences in host plant preference, a measure of habitat isolation among populations. These consistent differences among taxa highlight the important role of host plant use in promoting reproductive isolation and morphological variation among herbivorous insect populations–a prerequisite for ecological speciation.     

Admixture in Humans of Two Divergent Plasmodium knowlesi Populations Associated with Different Macaque Host Species

Human malaria parasite species were originally acquired from other primate hosts and subsequently became endemic, then spread throughout large parts of the world. A major zoonosis is now occurring with Plasmodium knowlesi from macaques in Southeast Asia, with a recent acceleration in numbers of reported cases particularly in Malaysia. To investigate the parasite population genetics, we developed sensitive and species-specific microsatellite genotyping protocols and applied these to analysis of samples from 10 sites covering a range of >1,600 km within which most cases have occurred. Genotypic analyses of 599 P. knowlesi infections (552 in humans and 47 in wild macaques) at 10 highly polymorphic loci provide radical new insights on the emergence. Parasites from sympatric long-tailed macaques (Macaca fascicularis) and pig-tailed macaques (M. nemestrina) were very highly differentiated (FST = 0.22, and K-means clustering confirmed two host-associated subpopulations). Approximately two thirds of human P. knowlesi infections were of the long-tailed macaque type (Cluster 1), and one third were of the pig-tailed-macaque type (Cluster 2), with relative proportions varying across the different sites. Among the samples from humans, there was significant indication of genetic isolation by geographical distance overall and within Cluster 1 alone. Across the different sites, the level of multi-locus linkage disequilibrium correlated with the degree of local admixture of the two different clusters. The widespread occurrence of both types of P. knowlesi in humans enhances the potential for parasite adaptation in this zoonotic system.     

两种亚热带榕属乔木植冠损伤后的位置依赖性枝发生

三峡库区需要大量植株用于三峡工程所涉及的诸多建设(如铁路、公路、建筑)之后的植被恢复和绿化。由于具有优美树冠和耐瘠薄土壤的能力,榕(Ficus microcarpa L.)和黄桷树(F. virens Ait. var. sublanceolata (Miq.) Cornor)在三峡库区作为绿化和行道树木大量栽植。在三峡库区,这两种树种苗木的培育主要通过切枝扦插的方式进行。大量切枝损伤植株植冠并且使叶组织数量减少,对植株生长有很大影响。植株生长与植株的枝发生格局有很大关系,为明确植冠损伤对植株生长的可能影响,对榕和黄桷树植冠损伤后的枝发生进行了研究。实验发现,切枝造成的植冠损伤对榕和黄桷树植株侧枝上的枝发生没有影响,不同损伤强度之间和不同损伤发生时间之间都不存在明显的差异。但是,植冠损伤后,榕和黄桷树植株主茎上的枝发生是位置依赖性的。植冠损伤不影响榕和黄桷树植株损伤处理后新生主茎段和具侧枝主茎段上的枝发生,却促进了损伤处理后裸露主茎段上的枝发生,并且裸露主茎段上枝发生的数量和密度随植冠损伤强度的升高而增大。此外,实验结果表明, 秋季植冠损伤植株裸露主茎段上的枝发生要高于春季植冠损伤植株裸露主茎段上的枝发生; 在榕和黄桷树的枝发生中,仅有裸露主茎段上增强的枝发生会促进植冠损伤植株对叶组织的生物量投     

两种热带雨林树苗对环境光强变化的生理响应和适应机制

干季末雨季初以西双版纳热带雨林中木奶果和玉蕊2种树苗为材料,研究了将生长于12.5%自然光（相当于小林窗的光强）和36%自然光（相当于大林窗的光强）下的这2种树苗分别移至36%自然光和12.5%自然光下之后各自叶片最大净光合速率（Pmax）、叶绿素荧光参数、光合色素含量、比叶重（LMA）以及叶片悬挂角（MA）的变化过程,探讨了2种植物幼苗在生长环境光强改变后其形态和生理生态特性做出的相应调整以适应新的光环境的过程与机制。结果表明,这2种树苗均不适宜生长在大林窗的强光环境下,但木奶果对光环境的增强表现出一定的耐受力,光合适应潜力强于玉蕊,2种树苗对低光环境都能较好地适应;新叶的生成在整株植物对生长光环境变化的适应过程中也起到至关重要的作用。     

SDS-PAGE Pattern Polymorphism of X-Chromosomal Glue Proteins in Natural Populations of Two Drosophila nasuta Subgroup Species

For the first time, the glue protein patternpolymorphism in natural populations of D. n.nasuta and D. s. neonasuta has beenanalyzed by SDS-PAGE. The study involving 200 and 185isofemale lines comprising 2028 and 1900 individuals of D. n.nasuta and D. s. neonasuta, respectively,revealed the occurrence of eight variant glue proteinphenotypes in D. n. nasuta and seven in D.s. neonasuta. The number and frequency of variant phenotypes in differentpopulations of both species were found to vary. Analysisof glue protein patterns in the F₁ progeny ofcrosses involving parents with variant glue proteinphenotypes revealed that the polymorphic fractions areproduced by co-dominant genes located on the Xchromosome. More than 87% of the naturally inseminatedadult females were found to produce polymorphic progeny. The heterozygous female larvae were found toexceed the homozygotes in the isofemale line progeny ofmost of the populations.     

Effects of Harvest on the Sustainability and Leaf Productivity of Populations of Two Palm Species in Maya Homegardens

Traditional management practices are usually thought to be sustainable. The Maya manage Sabal (Arecaceae) palms in homegardens, using their leaves for thatching. The sustainability of such production systems depends on the long-term persistence of palm populations, whereas resource availability also depends on the number of leaves on individual palms. We examined how leaf harvest affects Sabal yapa and S. mexicana population growth rates (λ) and leaf production, comparing traditional and alternative harvest regimes in terms of sustainability and productivity. Demographic, harvest and leaf production data were recorded for three years in two homegardens. We used general integral projection models linked to leaf-production models to describe population dynamics and productivity. Harvest had no effect on S. yapa’s vital rates or on λ, but it changed the growth rate of individuals of S. mexicana, with a negligible impact on λ. Homegardens affected λ values, reflecting the species’ ecological affinities. S. mexicana, introduced from mesic forests, required watering and shade; therefore, its population declined rapidly in the homegarden that lacked both water and shade. The λ of the xerophilic S. yapa was slightly larger without watering than with watering. Palms usually compensated for leaf extraction, causing the number of leaves harvested per individual to increase with harvest intensity. Nevertheless, traditional management is relatively mild, allowing standing leaves to accumulate but reducing the homegarden’s yield. Apparently, the Maya do not seek to maximize annual production but to ensure the availability of large numbers of leaves in homegardens. These leaves may then be used when the entire roof of a hut needs to be replaced every few years.     

Genetic Variation in Natural Populations of Five Drosophila Species and the Hypothesis of the Selective Neutrality of Protein Polymorphisms

We have studied genetic variation at 30-32 loci coding for enzymes in natural populations of five species of Drosophila. The average proportion of heterozygous loci per individual is 17.7 +/- 0.4%. The average proportion of polymorphic loci per population is 69.2 +/- 2.6% or 49.8 +/- 2.2%, depending on what criterion of polymorphism is used. The following generalizations are advanced: (1) The amount of genetic polymorphism varies considerably from locus to locus. (2) At a given locus, populations of the same species are very similar in the amount and pattern of genetic variation. (3) However, at some loci large differences sometimes occur between local populations of the same species. (4) The amount of variation at a given locus is approximately the same in all five species. (5) When different species are compared, the pattern of the variation is either essentially identical or totally different at a majority of loci. We have tested the hypothesis that protein polymorphisms are selectively neutral by examining four predictions derived from the hypothesis. Our results are at variance with every one of the predictions. We have measured the amount of genetic differentiation, D, between taxa of various degrees of evolutionary divergence. The average value of D is 0.033 for local populations, 0.228 for subspecies, 0.226 for semispecies, 0.538 for sibling species, and 1.214 for morphologically distinguishable species. Our results indicate that a substantial degree of genetic differentiation (22.8 allelic substitutions for every 100 loci) occurs between allopatric populations that have diverged to the point where they might become different species if they were to become sympatric. However, very little additional genetic change is required for the development of complete reproductive isolation. After the speciation process is completed, species continue to diverge genetically from each other.     

Changes in the Endosperm Cell Walls of Two Datura Species before Radicle Protrusion

The possibility of an association between changes in cell walls of the micropylar portion of the endosperm and the induction of germination was explored in seeds of Datura ferox and Datura stramonium. The structure of the inner surface of the endosperm was studied by scanning electron microscopy and the composition of cell wall polysaccharides analyzed by gas chromatography and gas chromatography-mass spectrometry. Both scanning electron microscope images and chemical analysis showed changes in the micropylar portion of the endosperm in induced seeds before radicle protrusion. The inner surface of the endosperm appeared eroded, and in some areas, wall material seemed to be missing. The content of the main component of the cell wall polysaccharides, containing predominantly 4-linked mannose, decreased well before the emergence of the radicle through the endosperm. We propose that the degradation of a mannan type polysaccharide is an important factor in the reduction in mechanical strength of the endosperm, thus facilitating germination.     

Protein Kinase Activity from Vaccinia Virions: Solubilization and Separation into Heat-Labile and Heat-Stable Components

A protein kinase was solubilized from whole vaccinia virions by using a solution containing deoxycholate, dithiothreitol, and sodium or potassium chloride. The released enzyme was completely dependent on Mg(2+) and was greatly stimulated by added basic proteins such as protamine or histones. Dithiothreitol was also stimulatory, whereas GTP, CTP, UTP, and P(i) at concentrations equimolar with ATP had little or no effect. Attempts to purify the protein kinase were initially unsuccessful, leading us to consider that either the enzyme was extremely labile or that two readily separable components were required for activity. The observation that the material extracted with NP-40 detergent during the preparation of viral cores stimulated the protein kinase activity of the intact cores supported the second possibility. As the protein kinase, now solubilized from viral cores, was passed through successive DEAE-cellulose columns, it became increasingly dependent for activity on addition of the NP-40 extract. A 30- to 40-fold stimulation of protein kinase activity, which afforded recovery of essentially all starting activity, could be effected by addition of the NP-40 extract to the partially purified enzyme. The NP-40 extract was shown to contain a heat stable, trypsin-sensitive protein, whose action could not be duplicated by cyclic nucleotides.     

冬油菜和春油菜在冷驯过程中膜脂分子的变化

研究了冬油菜和春油菜两种油菜品种在4℃冷驯3天后11类151种膜脂分子的变化,计算了其中膜脂的相对含量、双键指数和碳链长度值。结果发现油菜膜脂的膜脂分子组成模式及其冷驯诱导的膜脂分子组成变化与前期实验中的拟南芥情况非常相似,冷驯3天诱导的膜脂分子组成变化较小,但是膜脂的双键指数略有升高,春油菜双键指数和冷驯积累的脯氨酸含量都比冬油菜多。上述结果说明,油菜的冷驯需要更多的时间,春油菜对冷驯比较敏感。     

Contrasting Genetic Structure among Populations of Two Amphidromous Fish Species (Sicydiinae) in the Central West Pacific

Both present-day and past processes can shape connectivity of populations. Pleistocene vicariant events and dispersal have shaped the present distribution and connectivity patterns of aquatic species in the Indo-Pacific region. In particular, the processes that have shaped distribution of amphidromous goby species still remain unknown. Previous studies show that phylogeographic breaks are observed between populations in the Indian and Pacific Oceans where the shallow Sunda shelf constituted a geographical barrier to dispersal, or that the large spans of open ocean that isolate the Hawaiian or Polynesian Islands are also barriers for amphidromous species even though they have great dispersal capacity. Here we assess past and present genetic structure of populations of two amphidromous fish (gobies of the Sicydiinae) that are widely distributed in the Central West Pacific and which have similar pelagic larval durations. We analysed sections of mitochondrial COI, Cytb and nuclear Rhodospine genes in individuals sampled from different locations across their entire known range. Similar to other Sicydiinae fish, intraspecific mtDNA genetic diversity was high for all species (haplotype diversity between 0.9–0.96). Spatial analyses of genetic variation in Sicyopus zosterophorum demonstrated strong isolation across the Torres Strait, which was a geologically intermittent land barrier linking Australia to Papua New Guinea. There was a clear genetic break between the northwestern and the southwestern clusters in Si. zosterophorum (φ_ST = 0.67502 for COI) and coalescent analyses revealed that the two populations split at 306 Kyr BP (95% HPD 79–625 Kyr BP), which is consistent with a Pleistocene separation caused by the Torres Strait barrier. However, this geographical barrier did not seem to affect Sm. fehlmanni. Historical and demographic hypotheses are raised to explain the different patterns of population structure and distribution between these species. Strategies aiming to conserve amphidromous fish should consider the presence of cryptic evolutionary lineages to prevent stock depletion.