Saccharomyces cerevisiae-based molecular tool kit for manipulation of genes from gram-negative bacteria

A tool kit of vectors was designed to manipulate and express genes from a wide range of gram-negative species by using in vivo recombination. Saccharomyces cerevisiae can use its native recombination proteins to combine several amplicons in a single transformation step with high efficiency. We show that this technology is particularly useful for vector design. Shuttle, suicide, and expression vectors useful in a diverse group of bacteria are described and utilized. This report describes the use of these vectors to mutate clpX and clpP of the opportunistic pathogen Pseudomonas aeruginosa and to explore their roles in biofilm formation and surface motility. Complementation of the rhamnolipid biosynthetic gene rhlB is also described. Expression vectors are used for controlled expression of genes in two pseudomonad species. To demonstrate the facility of building complicated constructs with this technique, the recombination of four PCR-generated amplicons in a single step at >80% efficiency into one of these vectors is shown. These tools can be used for genetic studies of pseudomonads and many other gram-negative bacteria.     

Hot topic or hot air? Climate change and malaria resurgence in East African highlands

Climate has a significant impact on malaria incidence and we have predicted that forecast climate changes might cause some modifications to the present global distribution of malaria close to its present boundaries. However, it is quite another matter to attribute recent resurgences of malaria in the highlands of East Africa to climate change. Analyses of malaria time-series at such sites have shown that malaria incidence has increased in the absence of co-varying changes in climate. We find the widespread increase in resistance of the malaria parasite to drugs and the decrease in vector control activities to be more likely driving forces behind the malaria resurgence.     

The Sec1/Munc18 Protein Vps45 Regulates Cellular Levels of Its SNARE Binding Partners Tlg2 and Snc2 in Saccharomyces cerevisiae

Intracellular membrane trafficking pathways must be tightly regulated to ensure proper functioning of all eukaryotic cells. Central to membrane trafficking is the formation of specific SNARE (soluble N-ethylmeleimide-sensitive factor attachment protein receptor) complexes between proteins on opposing lipid bilayers. The Sec1/Munc18 (SM) family of proteins play an essential role in SNARE-mediated membrane fusion, and like the SNAREs are conserved through evolution from yeast to humans. The SM protein Vps45 is required for the formation of yeast endosomal SNARE complexes and is thus essential for traffic through the endosomal system. Here we report that, in addition to its role in regulating SNARE complex assembly, Vps45 regulates cellular levels of its SNARE binding partners: the syntaxin Tlg2 and the v-SNARE Snc2: Cells lacking Vps45 have reduced cellular levels of Tlg2 and Snc2; and elevation of Vps45 levels results in concomitant increases in the levels of both Tlg2 and Snc2. As well as regulating traffic through the endosomal system, the Snc v-SNAREs are also required for exocytosis. Unlike most vps mutants, cells lacking Vps45 display multiple growth phenotypes. Here we report that these can be reversed by selectively restoring Snc2 levels in vps45 mutant cells. Our data indicate that as well as functioning as part of the machinery that controls SNARE complex assembly, Vps45 also plays a key role in determining the levels of its cognate SNARE proteins; another key factor in regulation of membrane traffic.     

血管内皮祖细胞与糖尿病微血管病变研究进展

糖尿病微血管病变严重影响了患者生活质量,是患者致死致残主要原因。微血管病变主要表现在视网膜、肾、神经、心肌组织。微血管病变的机制尚未完全清楚,近年越来越多研究发现血管内皮祖细胞(endothelial progenitor cells,EPCs)是该病发病重要原因。EPCs有分化为成熟的内皮细胞并且参与新血管形成和新生的能力。正常情况下内皮损失和EPCs对内皮的修复作用处于动态平衡状态,一旦EPCs受损,内皮损害和修复之间的平衡被打破,内皮层的完整性遭到破坏,必然参与糖尿病血管病变的发生发展。国内外大量研究证明糖尿病合并大血管病变EPCs数目功能改变,而糖尿病合并微血管病变EPCs的怎样变化?本文就EPCs与糖尿病微血管病变的关系进行系统综述。     

Stage-specificity of spontaneous mutation at a tandem repeat DNA locus in the mouse germline

Mouse expanded simple tandem repeat (ESTR) loci are the most unstable loci in the mouse genome. Despite the fact that over the last decade these loci have been extensively used for studying germline mutation induction in mice, to date little is known about the mechanisms underlying spontaneous and induced ESTR mutation. Here we used flow cytometry and single-molecule PCR to compare the frequency of ESTR mutation in four flow-sorted fractions of the mouse male germ cells – spermatogonia, spermatocytes I, round and elongated spermatids. The frequency and the spectrum of ESTR mutation did not significantly differ between different stages of mouse spermatogenesis. Considering these data and the results of other publications, we propose that spontaneous ESTR mutation is mostly attributed to replication slippage in spermatogonia and these loci may be regarded as a class of expanded microsatellites.     

Cellulose conversion in dry grind ethanol plants

The expansion of the dry grind ethanol industry provides a unique opportunity to introduce cellulose conversion technology to existing grain to ethanol plants, while enhancing ethanol yields by up to 14%, and decreasing the volume while increasing protein content of distiller's grains. The technologies required are cellulose pretreatment, enzyme hydrolysis, fermentation, and drying. Laboratory data combined with compositional analysis and process simulations are used to present a comparative analysis of a dry grind process to a process with pretreatment and hydrolysis of cellulose in distiller's grains. The additional processing steps are projected to give a 32% increase in net present value if process modifications are made to a 100 million gallon/year plant.     

Topographically generated fronts, very nearshore oceanography and the distribution and settlement of mussel larvae and barnacle cyprids

Flow patterns adjacent to shore may prevent or aid shorewardmigration of benthic invertebrate larvae. We hypothesized thata front at the mouth of Sunset Bay, Oregon, prevents shorewarddispersal of larvae, significantly altering settlement of mussellarvae and barnacle cyprids. Settlement was measured at threesets of moorings (three moorings per site) distributed acrossthe front at Sunset Bay. From 6 July to 4 September 2000, sampleswere collected roughly every other day. Concurrently, we madevertical zooplankton tows adjacent to each mooring site andcollected physical oceanographic data. During upwelling-favorablewinds, the front was always present at the bay mouth, separatingsignificantly cooler, saltier and denser offshore water fromthat within the bay. During downwelling winds, the front brokedown and we found no significant difference in the surface physicaloceanographic parameters across the bay mouth. During upwelling,the concentration of mussel larvae was higher seaward of thefront than landward, but there was no significant differencein concentration during downwelling, suggesting that the frontmay act as a barrier to the shoreward dispersal of mussels.Mussel settlement was too low and sporadic to allow statisticalanalysis. There was no difference in cyprid concentrations acrossthe bay mouth whether the front was present or not. Cyprid settlementwas, however, nearly an order of magnitude lower at mooringsseaward of the front than at those landward. A significant cross-correlationwas found between settlement at the offshore mooring and tidalrange (r = 0.464, lag = 0 days) and between settlement at themid and inner moorings and downwelling winds (r = 0.532 midbay, r = 0.532 inner bay, lag = 0 days). Seaward of the front,settlement varied with tidal range, while landward of the front,most settlement occurred as brief pulses during downwellingwinds, periods when the front was not present. We found largedifferences in the distribution of cyprids, and mussel larvaeand cyprid settlement relative to the front; larval distributionsand settlement varied with upwelling versus downwelling windsand was due to differences in the very nearshore (i.e. within100–1000 m of shore) coastal oceanography.     

Observations on the distribution of meroplankton during an upwelling event

The distribution of the larvae of benthic invertebrates wasinvestigated relative to hydrographic structures as a test ofthe hypothesis that larvae behave as if they are passive particles.Observations of larval and oceanographic distributions weremade off Duck, North Carolina, USA in August 1994. Conditionswere characterized by wind-driven coastal upwelling; flow wasgenerally offshore near the surface and onshore below the pycnocline.Within 5 km of the shore the pycnocline was bent upward by theupwelling and it intersected the surface along most of the transects.In zooplankton samples, 20 taxa of larvae were counted (10 bivalveveligers, nine gastropod veligers and one polychaete larvae).Using cluster analysis, larvae were separated into groups withsimilar patterns of distribution and similar affinities to waterproperties. The larvae in Cluster 3 did not display a consistentdistribution pattern beyond that they tended to be found inwarmer surface waters. An earlier paper described the distributionof larvae in the same location during a downwelling event [A.Shanks et al. (2002) J. Plankton Res., 24, 391–416]. Twoof the clusters identified during this previous study were quitesimilar in composition to Clusters 1 and 2 in this study. Inboth studies, Cluster 1 larvae were found below the pycnocline,but during the upwelling event they were transported shorewardwith the advection of the subpycnocline waters by the upwellingcirculation. Within 5 km of the shore, Cluster 1 larvae werefound at depths shallower than the base of the pycnocline andwere often found in patches of high larval concentration. Thepatches were located where the waters were upwelling. Cluster2 larvae were found within 5 km of the shore in both studiesand tended to be highly concentrated in convergences or divergences.Larvae in Cluster 1 generally appeared to be dispersing as passiveparticles, except within the zone of upwelling where they mayhave been swimming against the upwelling flow leading to higherlarval concentrations. Cluster 2 larvae appeared to be consistentlyconcentrated in areas of vertical currents, suggesting thatthey may be attempting to maintain a preferred depth in theface of the vertical flow which would lead to high larval concentrationand nearshore larval distributions despite extensive cross-shelfmovement of water. Despite their slow swimming speeds, the larvaein Clusters 1 and 2 were not swept offshore by the upwellingevent.     

Previous agonistic experience determines both foraging behavior and territoriality in the limpet Lottia gigantea (Sowerby)

Lottia gigantea, the owl limpet, is an algal gardener. Territorial individuals actively defend gardens, which consist of clearedareas in the intertidal zone upon which a thick algal filmdevelops and upon which the territory holder feeds. Smaller,nonterritory holders raid these gardens and graze the algalfilm. Territorial individuals must obtain an adequate ration without compromising the productivity of the garden. In contrast,a nonterritory holder grazing on another limpet's territorymust obtain an adequate ration before it contacts the territoryholder and is driven off. In the laboratory, replicate setsof 10 limpets were trained to behave territorially and nonterritorially.Training mimicked natural encounters between territorial andnonterritorial L. gigantea. Limpets given territorial trainingleft significantly (t = -4.92, df = 9, p =.00041) more algalcover behind when grazing (on average 71%) than did limpetstrained to be nonterritorial (on average 50%). Territorial limpets seldom grazed over the same area more than once (4% of the grazedarea). In contrast, nonterritorial limpets frequently foragedover an area more than once; of the area grazed, 20% had beenvisited more than once. Previous agonistic experience determinesboth territorial behavior and foraging strategies, two of thecritical behaviors necessary for successful gardening behavior.Nonterritorial limpets maximize consumption per unit area, whereas territorial limpets appear to forage prudently, leaving a significantly greater proportion of the plant biomass behind.