Coated-vesicle formation in vitro: conflicting results using different assays

Cell-free systems provide essential tools for elucidating the molecular mechanisms underlying complex cellular processes such as vesicular transport. The biochemical utility of these model systems is strengthened by assays that allow rapid, quantitative detection of the events being studied. Two model systems have recently been developed to reconstitute coated-vesicle budding, and two different biochemical assays are used to detect this event. Striking differences in the biochemical requirements for 'coated-vesicle budding' are detected by these two assays, suggesting that two distinct events are being measured. These findings have wide implications for the use of cell-free assay systems in cell biology.     

Molecular diversity of sulfate-reducing bacteria from two different continental margin habitats

This study examined the natural diversity and distributions of sulfate-reducing bacteria along a natural carbon gradient extending down the shelf-slope transition zone of the eastern Pacific continental margin. Dissimilatory (bi)sulfite reductase gene sequences (dsrAB) were PCR amplified and cloned from five different sampling sites, each at a discrete depth, from two different margin systems, one off the Pacific coast of Mexico and another off the coast of Washington State. A total of 1,762 clones were recovered and evaluated by restriction fragment length polymorphism (RFLP) analysis. The majority of the gene sequences recovered showed site and depth restricted distributions; however, a limited number of gene sequences were widely distributed within and between the margin systems. Cluster analysis identified 175 unique RFLP patterns, and nucleotide sequences were determined for corresponding clones. Several different continental margin DsrA sequences clustered with those from formally characterized taxa belonging to the delta subdivision of the class Proteobacteria (Desulfobulbus propionicus, Desulfosarcina variabilis) and the Bacillus-Clostridium (Desulfotomaculum putei) divisions, although the majority of the recovered sequences were phylogenetically divergent relative to all of the other DsrA sequences available for comparison. This study revealed extensive new genetic diversity among sulfate-reducing bacteria in continental margin sedimentary habitats, which appears to be tightly coupled to slope depth, specifically carbon bioavailability.     

Contradictory results from different methods for measuring direction of insect flight

1. Stream ecologists have been puzzled by the apparent paradox that invertebrate populations persist in headwater streams despite the high frequency with which individuals drift downstream. To resolve this ‘drift paradox’, directions and distances of both larval and adult movement must be identified. Using over 50 interception traps in combination with results from several mark–capture experiments using ¹⁵N as a label, we tested the assumption that interception traps accurately represent the ultimate direction of adult insect flight. 2. In several streams in the Hubbard Brook Experimental Forest, 76% of ¹⁵N‐labelled stoneflies (Leuctra ferruginea) had flown upstream from where they emerged to where they were captured. In contrast, over 60% of stoneflies were flying downstream when captured, i.e. on the upstream side of an interception trap. 3. The instantaneous direction, as indicated by the side of the interception trap on which they were captured, indicated the ultimate flight direction for fewer than 1/3 of the individuals captured. Thus, such traps did not accurately reflect the ultimate flight patterns of individuals, as indicated by mark–capture data. 4. Conclusions drawn from interception trap counts regarding the direction of movement and the distribution and persistence of populations may need to be re‐evaluated. We suggest that better tracking methods, including mass mark–capture studies using stable isotopes, be used to evaluate the potentially complex patterns of adult insect movement and the consequences of that movement for individuals and populations.     

Life span of B lymphocytes: the experimental basis for conflicting results

Recent claims have challenged the view that most peripheral, mature B cells are long-lived, and propose rates of peripheral decay that are compatible with bone marrow production. This disagreement can only reflect differences in the protocols and methods used to measure peripheral lymphocyte life spans. We have now assessed toxic or other nonselective effects of hydroxyurea treatment on the survival and migration of peripheral, noncycling cells, as well as possible reasons for exaggerated decays of LPS-reactive B cells transferred to LPS nonresponder hosts, the two methods leading to conclusions of short life spans. We also studied general effects on cell survival introduced by either repeated [3H]thymidine injections or the stress associated with surgery, thoracic duct cannulation in particular--methods with which the notion of long life spans had been established. The results failed to show toxic or nonselective effects of hydroxyurea treatments and artificial decays of LPS-reactive cells in adoptive hosts. In contrast, the present experiments demonstrate that both the stress associated with surgery and repeated [3H] thymidine administration profoundly deplete a pool of short-lived B cells, consequently selecting for an apparent higher proportion of long-lived cells.     

Dispersal between shallow and abyssal seas and evolutionary loss and regain of compound eyes in cylindroleberidid ostracods: conflicting conclusions from different comparative methods

Complex organs such as eyes are commonly lost during evolution, but the timescale on which lost phenotypes could be reactivated is a matter of long-standing debate, with important implications for the molecular mechanisms of trait loss. Two phylogenetic approaches have been used to test whether regain of traits has occurred. One way is by comparison of nested, continuous-time Markov models of trait evolution, approaches that we term tree-based tests. A second way to demonstrate statistical support for trait regain is through use of node-based tests that employ explicit estimation of ancestral node states. Here, we estimate new molecular and morphological phylogenies and use them to examine the possibility of eye regain and dispersal between abyssal and shallow seas during the history of cylindroleberidid ostracods, a family of about 200 species, comprising both eyeless and sighted species. First, we confirmed that eye presence/absence is correlated with habitat depth. Parameter estimates from a phylogenetic model indicate that speciation is more rapid in deep-sea eyeless clades compared with shallow-water sighted clades. In addition, we found that tree-based statistical tests usually indicated reversals, including both transitions from deep to shallow seas and regain of eyes. In contrast, node-based statistical tests usually failed to show significant support for reversals. These results also hold for simulated phylogenies, indicating that they are not unique to the current data set. We recommend that both tree-based and node-based tests should be examined before making conclusions about character reversal and that ideally, alternative character histories should be tested using additional data, besides just the phylogenetic distribution of presence/absence of the characters.     

Comparative evaluation of different methods for typing bacteria in the genus Proteus

The work presents the comparative evaluation of different methods used for differentiating bacteria of the genus Proteus. A scheme of the combined typing of Proteus is presented. This scheme includes the determination of enzymatic and serological variants with the subsequent additional bacteriocinogenic and phage typing. Among the Proteus strains in our collection (600 strains), 123 sero-enzymatic variants, 71 enzymo-lysogenic variants, 64 enzymo-bacteriocinogenic variants, 162 sero-lysogenic variants, 164 sero-bacteriocinogenic variants and 52 bacteriocinogeno-lysogenic variants have been detected.     

Studies on psychrophilic bacteria in two lakes of different trophy

The number of bacteria capable of growth at low temperatures in two lakes was found to be subject to considerable variation throughout the year although the changes were not correlated with changes in ambient temperature. A correlation was, however, observed between temperature and ratio of number of psychrophiles to "total" count. All of the psychrophilic bacteria isolated from the studied lakes were able to grow at temperatures below 0 degrees C. Microorganisms isolated from eutrophic lakes were characterized by more pronounced stenothermy than those isolated from lake with low tropic level. These bacteria represented a type resembling a type resembling obligatory or ,,true" psychrophiles. Most of the isolates belonged to the genus Pseudomonas and to the Flavobacterium--Cytophaga group.     

Evaluation of different methods to discriminate Bacillus anthracis from other bacteria of the Bacillus cereus group

Aims:  To evaluate different methods that are useful for rapid and definitive discrimination of Bacillus anthracis from other bacteria of the Bacillus cereus group in environmental samples like letters claimed to contain anthrax spores.
Methods and Results:  Characterized strains and bacteria from environmental samples were analysed by microbiological and molecular methods (PCR and restriction analysis). Environmental isolates often shared several microbiological features with B. anthracis , e.g. lack of β -haemolysis and phospholipase C activity, and only the gamma phage assay was specific for B. anthracis . PCR assays targeting markers from the virulence plasmids exclusively detected B. anthracis , but other PCR targets were also detected in nonanthrax isolates. Additionally, the restriction pattern in an Alu I restriction analysis of the SG-749 fragment is not 100% specific. The loci used for multiple-locus variable-number tandem repeat analysis of B. anthracis are also present in other members of the B. cereus group, but amplicon sizes are usually different.
Conclusions:  Environmental samples often contain borderline isolates closely related to B. anthracis both on microbiological and genetic levels. Real-time PCR targeting plasmidal and chromosomal markers should be used for rapid and definitive exclusion of a virulent strain of B. anthracis in such samples.
Significance and Impact of the Study:  This study gives an overview of the current microbiological and molecular methods used for identification of B. anthracis and shows that most assays have limits when borderline isolates present in environmental samples are analysed.     

A comparison between two methods of extracting chlorophyll-a from different phytoplankton samples

Summary A comparison has been made between chlorophyll-a estimations based on the cold acetone extraction procedure and on the hot ethanol extraction method. When comparing the results obtained with both methods a standard deviation of 20% must be taken into account. A correlation between extraction efficiency and phytoplankton species composition could not be found. Although the chlorophyll-a yield in ethanol extracts is not better than that in acetone extracts, there are several advantages in using the ethanol method, particularly the short extraction period and the cheaper procedure.     

Effect of different allocation methods on LCA results of products from wild-caught fish and on the use of such results

Purpose  

The purpose of this study has been to investigate the effect of different allocation methods on life cycle assessment (LCA) results of products derived from line-caught cod and the consequences of applying these methods considering the main aims of this case study. These aims were for internal improvement work and communication of results to the market.     

两种不同方法对阴道感染的评价

目的探讨需氧菌阴道炎／细菌性阴道病五项联合测定试剂对阴道感染的临床应用价值。方法将380例就诊女性采用需氧菌阴道炎／细菌性阴道病五项联合测定试剂方法,同时采刖自带常规镜检＋PH值方法进行检测。结果380例阴道分泌物标本经五项联合测定试剂方法检出阴道微生态失衡345例（其中包括AV、BV、CV、VVT等感染）,阳性率为90．79％;采用白带常规镜检＋pH值方法检出阴道微生态失衡328例（其中包括AV、BV、CV、VVT等感染）,阳性率为86．32％,两种检查方法结果差异明显。结论需氧菌阴道炎／细菌性阴道病五项联合测定试剂法准确,快速,高效,能及时评价阴道微生态状况,提高临床合理用药程度,值得临床推广应用。     

Selection at the amino acid level can influence synonymous codon usage: implications for the study of codon adaptation in plastid genes

A previously employed method that uses the composition of noncoding DNA as the basis of a test for selection between synonymous codons in plastid genes is reevaluated. The test requires the assumption that in the absence of selective differences between synonymous codons the composition of silent sites in coding sequences will match the composition of noncoding sites. It is demonstrated here that this assumption is not necessarily true and, more generally, that using compositional properties to draw inferences about selection on silent changes in coding sequences is much more problematic than commonly assumed. This is so because selection on nonsynonymous changes can influence the composition of synonymous sites (i.e., codon usage) in a complex manner, meaning that the composition biases of different silent sites, including neutral noncoding DNA, are not comparable. These findings also draw into question the commonly utilized method of investigating how selection to increase translation accuracy influences codon usage. The work then focuses on implications for studies that assess codon adaptation, which is selection on codon usage to enhance translation rate, in plastid genes. A new test that does not require the use of noncoding DNA is proposed and applied. The results of this test suggest that far fewer plastid genes display codon adaptation than previously thought.     

Psychobiological consequences of two different weaning methods in sheep.

Weaning is associated with a break in the mother-infant contact. In sheep, under natural conditions, the rupture of the social bond is progressive. In contrast, weaning imposed by breeders may result in psychobiological disturbances. The aim of this experiment was to measure the behavioural, hormonal and immune consequences of two types of sudden weaning: total separation (TS) and partial separation (PS), in 42 Ile-de-France ewes and their 60 lambs. TS animals (lambs and ewes) vocalized less frequently and for a shorter period of time than did the PS animals. Neither the plasmatic cortisol level in lambs nor the humoral response of dams and young were modified by the two weaning methods. In contrast, the excretion of coccidial oocysts increased significantly after weaning in TS lambs only and the growth rate of TS females was lower than that of the other lambs. These results suggest that these methods of rupture of mother-young social contact have only limited negative consequences on both partners in sheep, contrary to primates and rodents.     

Cluster analysis of the codon use frequency of MHC genes from different species

The relative synonymous codon use frequency of 135 MHC genes from four mammal species (Homo sapiens, Pan troglodyte, Macaca mulanta and Rattus norvegicus) is analyzed using a hierarchical cluster method. The result suggests that gene function is the dominant factor that determines codon usage bias, while species is a minor factor that determines further difference in codon usage bias for genes with similar functions. The conclusion may be useful in gene classification and gene function prediction.     

Comparative investigation of different methods of storage of lactic acid bacteria

The study was undertaken to elucidate how different methods of storage (immersing in mineral oil, lyophilization, and subculturing) of lactic acid bacteria belonging to the generaLactobacillus andLactococcus affect their viability, antibiotic activity, and ability to accumulate organic acids. Storage of the lactic acid bacteriumLactococcus lactis subsp.lactis by immersion in mineral oil proved to be ineffective. Lyophilization allowed the survival of a sufficiently large number of cells, although their antibiotic activity somewhat decreased. The resuscitation of lyophilized bacteria by subculturing them in rich nutrient media, such as skim milk, led to the restoration of their physiological activity, including the effective antimicrobial spectrum     

Measure of synonymous codon usage diversity among genes in bacteria

Background  

In many bacteria, intragenomic diversity in synonymous codon usage among genes has been reported. However, no quantitative attempt has been made to compare the diversity levels among different genomes. Here, we introduce a mean dissimilarity-based index (Dmean) for quantifying the level of diversity in synonymous codon usage among all genes within a genome.     

Variation of RAPD-fingerprint patterns using different DNA-extraction methods with Gram-positive bacteria

The effect of the DNA-extraction method used on fingerprint patterns of RAPD was studied usingStaphylococcus epidermidis andStreptococcus faecalis. The three methods tested (Chelex, microwave and phenol/chloroform) led to significantly different RAPD patterns. The microwave technique generated reproducible patterns and seems the most suitable for RAPD analysis of Gram-positive bacteria.The authors are with the Laboratoire de Microbiologie, Hôpital Salvator, 249 bld Sainte Marguerite, 13009 Marseille, France     

A comparison of two methods and different media for isolatingFrankia from Casuarina root nodules

Four species of Casuarina were raised in the glasshouse and inoculated with nodules collected from nine different geographical areas within Australia. Isolations ofFrankia were attempted from 10 of the Casuarina-Frankia nodule combinations using two methods, a nodule dissection and a filtration method. With both techniquesFrankia isolates were obtained from four of the 10Frankia sources. Spores were not observed in sections of nodules from the four sources from whichFrankia was isolated, whereas spores were observed in the remaining six nodule sources. For selected nodule sources a range of isolation media were tried, but no improvement in the isolation success rate was achieved. The effect of host species on ease of isolation was studied. The results obtained suggested it was theFrankia strain and not the host plant species which determined the ease of isolation from Casuarina nodules.