IS2-43 and IS2-44: New alleles of the insertion sequence IS2 which have promoter activity

Summary The sequence of two new IS2 alleles with promoter activity (IS2-43 and IS2-44) is reported. The alleles are identical and are formed by a 17 bp tandem duplication in an AT-rich region of IS2. This created a new RNA polymerase binding site. A mutation was found that increased the frequency of formation of these 17 bp duplications but not of another class of duplications, the mini-insertions. This suggested that the mechanisms of formation of the two classes of duplications are different.     

Evaluation of a new model system for studying the formation of heterocyclic amines

Heterocyclic amines (HAs) are an important class of food mutagens and carcinogens, which can be found in cooked meat and fish. Increasing heating temperatures and times usually increase mutagenic activity in meat and meat extracts during cooking. We developed a model system, which allows to examine the effects of precursor composition and heating conditions (time and temperature) on the formation of HAs in meat. Homogenized and freeze dried meat samples (beef, pork chops, chicken breast and turkey breast) are heated with diethylene glycol in closed vials under stirring in a thermostated heating block. After an appropriate sample preparation (extraction and clean-up) ten different HAs were measured by HPLC analyses with gradient elution and mass selective detection. The time courses of HA-formation in the different kinds of meat at varying heating temperatures were determined up to heating times of 30 min. 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) was the most abundant HA in these experiments and reached the highest concentrations in the beef meat samples, as did the other HAs (MeIQ, AalphaC) at 220 degrees C in the heating block under stirred conditions. Additionally the influence of the antioxidant TBHQ (t-butylhydroquinone) on the formation of HAs in the model system was tested. However TBHQ effected only slight reductions of HA formation in all kinds of meat.     

Experimental murine amyloidosis: a model system for studying amyloid formation.

Myeloma-associated and casein-induced murine amyloidosis were used as models to study the role of lymphocytes and macrophages in amyloid formation. Amyloidosis occurred rarely and in small amounts in Balb/C mice with immunoglobulin (Ig)-producing myeloma tumours but large amounts could be induced by injections of casein. Fluorescent staining of both forms of amyloid deposits by means of anti-casein- and anti-myeloma-amyloid antibodies indicated that they either crossreacted or coexisted. Nor abnormality of Ig biosynthesis was detected in amyloidosis, suggesting that abnormal degradation was responsible for production of the Ig form of amyloid. Although spleen lymphocytes of casein-injected mice with amyloidosis demonstrated diminished cellular immunologic responses, this did not indicate generalized immunologic incompetence. The non-Ig form of amyloid in casein-injected mice was shown to be produced by macrophages, and a technique was developed for increasing the yield of amyloid-containing cells.     

Potassium release, a useful tool for studying antimicrobial peptides

We used an ion-selective electrode to measure potassium release from bacteria treated with antimicrobial peptides. This broadly applicable and simple technique for assessing membrane integrity deserves greater use.     

The continuum concept remains a useful framework for studying mycorrhizal functioning

Background

Recent studies have questioned the validity of the mutualism-parasitism continuum of mycorrhizal function. This paper re-evaluates the continuum model and analyzes these concerns.

Scope and Conclusions

Three insights arise from this analysis. First, the continuum model defines mycorrhizal function as an emergent property of complex interactions. The model identifies resource trade and symbiotic control as key determinants of the costs and benefits of the symbiosis for plants and fungi, and the interaction of these factors with the environment ultimately controls mycorrhizal function. Second, analysis of carbon costs and phosphorus benefits is too narrow a focus to accurately predict mycorrhizal function. Analysis of plant and fungal fitness responses in ecologically and evolutionarily relevant systems are required to elucidate the full range of nutritional and non-nutritional factors embodied within mycorrhizal functioning. Finally, the definition of the term ‘parasitism’ has evolved. Some fields of science maintain the original definition of a nutritional relationship between host and parasite while other fields define it as a +/- fitness relationship. This has generated debate about whether the continuum of mycorrhizal functioning should properly be called a positive–negative response continuum or a mutualism-parasitism continuum. This controversy about semantics should be resolved, but it does not overturn the continuum concept.     

Beta-subunit of ATP-synthase: a useful marker for studying the phylogenetic relationship of eubacteria

The genes encoding the beta-subunits of ATP-synthases (ATPases) from Bacteroides fragilis DSM 2151, Cytophaga lytica DSM 2039 and 'Taxeobacter ocellatus' were cloned. The nucleotide sequences were determined completely for the genes of the first two organisms and to a major part for that of 'T. ocellatus'. The predicted amino acid sequences were compared with previously published amino acid sequences of beta-subunits. Two characteristic insertions were found in genes from organisms belonging to the so-called bacteroides-cytophaga-flavo-bacterium group. The remaining structure shows a high degree of sequence similarity within this group. These data support the conclusions drawn from comparative 16S rRNA sequence analyses that organisms in this phenotypically heterogeneous group are phylogenetically related. A phylogenetic tree was constructed based on a distance matrix of optimally aligned amino acid sequences of beta-subunits of ATPases of various eubacteria, chloroplasts and mitochondria. It is in good agreement with a tree derived from 16S rRNA sequence analyses.     

Doubly uniparental inheritance of mitochondria as a model system for studying germ line formation

Background

Doubly Uniparental Inheritance (DUI) of mitochondria occurs when both mothers and fathers are capable of transmitting mitochondria to their offspring, in contrast to the typical Strictly Maternal Inheritance (SMI). DUI was found in some bivalve molluscs, in which two mitochondrial genomes are inherited, one through eggs, the other through sperm. During male embryo development, spermatozoon mitochondria aggregate in proximity of the first cleavage furrow and end up in the primordial germ cells, while they are dispersed in female embryos.

Methodology/Principal Findings

We used MitoTracker, microtubule staining and transmission electron microscopy to examine the mechanisms of this unusual distribution of sperm mitochondria in the DUI species Ruditapes philippinarum. Our results suggest that in male embryos the midbody deriving from the mitotic spindle of the first division concurs in positioning the aggregate of sperm mitochondria. Furthermore, an immunocytochemical analysis showed that the germ line determinant Vasa segregates close to the first cleavage furrow.

Conclusions/Significance

In DUI male embryos, spermatozoon mitochondria aggregate in a stable area on the animal-vegetal axis: in organisms with spiral segmentation this zone is not involved in cleavage, so the aggregation is maintained. Moreover, sperm mitochondria reach the same embryonic area in which also germ plasm is transferred. In 2-blastomere embryos, the segregation of sperm mitochondria in the same region with Vasa suggests their contribution in male germ line formation. In DUI male embryos, M-type mitochondria must be recognized by egg factors to be actively transferred in the germ line, where they become dominant replacing the Balbiani body mitochondria. The typical features of germ line assembly point to a common biological mechanism shared by DUI and SMI organisms. Although the molecular dynamics of the segregation of sperm mitochondria in DUI species are unknown, they could be a variation of the mechanism regulating the mitochondrial bottleneck in all metazoans.     

Somatic mutations as a useful tool for studying clonal dynamics in trees

The seemingly eternal cycles of clonal growth in many tree species, with members of Populus (aspen, poplars, cottonwoods and the like) featuring most prominently, provoke a number of questions on the interface between ecology, genetics and forestry. In this issue, two groups present their approaches to clonal dynamics ( Ally et al. 2008 and Mock et al. 2008 ), using microsatellite (or simple sequence repeat, SSR) variation in P. tremuloides. Ally et al. developed and applied a model for using microsatellites to estimate clone age and infer other community characteristics. Mock et al. used fewer microsatellites but in more individuals, to examine clone size and distribution across the landscape.     

Viroid: a useful model for studying the basic principles of infection and RNA biology

Viroids are small, circular, noncoding RNAs that currently are known to infect only plants. They also are the smallest self-replicating genetic units known. Without encoding proteins and requirement for helper viruses, these small RNAs contain all the information necessary to mediate intracellular trafficking and localization, replication, systemic trafficking, and pathogenicity. All or most of these functions likely result from direct interactions between distinct viroid RNA structural motifs and their cognate cellular factors. In this review, we discuss current knowledge of these RNA motifs and cellular factors. An emerging theme is that the structural simplicity, functional versatility, and experimental tractability of viroid RNAs make viroid-host interactions an excellent model to investigate the basic principles of infection and further the general mechanisms of RNA-templated replication, intracellular and intercellular RNA trafficking, and RNA-based regulation of gene expression. We anticipate that significant advances in understanding viroid-host interactions will be achieved through multifaceted secondary and tertiary RNA structural analyses in conjunction with genetic, biochemical, cellular, and molecular tools to characterize the RNA motifs and cellular factors associated with the processes leading to systemic infection.     

Colicin import and pore formation: a system for studying protein transport across membranes?

Pore-forming colicins are a family of protein toxins (M_r40–70kDa) produced by Escherichia coli and related bacteria. They are bactericidal by virtue of their ability to form ion channels in the inner membrane of target cells. They provide a useful means of studying questions such as toxin action, polypeptide translocation across and into membranes, voltage-gated channels and receptor function. These colicins bind to a receptor in the outer membrane before being translocated across the cell envelope with the aid of helper proteins that belong to nutrient-uptake systems and the so-called‘Tol’proteins, the function of which has not yet been properly defined. A distinct domain appears to be associated with each of three steps (receptor binding, translocation and formation of voltage-gated channels). The Tol-dependent uptake pathway is described here. The structures and interactions of TolA, B, Q and R have by now been quite clearly defined. Transmembrane α-helix interactions are required for the functional assembly of the E. coli Tol complex, which is preferentially located at contact sites between the inner and outer membranes. The number of colicin translocation sites is about 1000 per cell. The role and the involvement of the OmpF porin (with colicins A and N) have been described in a recent study on the structural and functional interactions of a colicin-resistant mutant of OmpF. The X-ray crystal structure of the channel-forming fragment of colicin A and that of the entire colicin la have provided the basis for biophysical and site-directed muta-genesis studies. Thanks to this powerful combination, it has been established that the interaction with the receptor in the outer membrane leads to a very substantial conformational change, as a result of which the N-terminal domains of colicins interact with the lumen of the OmpF pore and then with the C-terminal domain of TolA. A molten globular conformation of colicins probably constitutes the intermediate translocation/insertion competent state. Once the pore has formed, the polypeptide chain spans the whole cell envelope. Three distinct steps occur in the last stage of the process: (i) fast binding of the C-terminal domain to the outer face of the cytoplasmic membrane; (ii) a slow insertion of the polypeptide chain into the outer face of the inner membrane in the absence of Δψ and (iii) a profound reorganization of the helix association, triggered by the transmembrane potential and resulting in the formation of the colicin channel.     

Bacterial adherence to hydrocarbons: a useful technique for studying cell surface hydrophobicity

Abstract Bacterial adherence to hydrocarbons (BATH) is a simple and rapid technique for determining cell-surface hydrophobicity. During recent years, this method has found application in the study of the surface characteristics of a wide variety of bacteria and bacterial mixtures. Correlations have been found between the adherence of bacteria to hydrocarbons and their attachment to other surfaces, including non-wettable plastics, epithelial cells, and teeth. A slight modification of the assay enables the isolation of nonhydrophobic mutants. The present publication briefly describes the technique and its modifications, summarizes results obtained using this method, and suggests several directions for further investigation.     

Progressive hypoxia until brain electrical silence: a useful model for studying protective interventions

Anesthetized spontaneously breathing rats, fitted with epicortical electrodes and catheters for sampling arterial, venous, and cerebral venous blood, were exposed to standardized progressive hypoxia. Three minutes of hypoxia sequentially caused hyperpnea, hypopnea, apnea, and cessation of electrocorticogram "spiking," of synchronization, and of background in electroencephalogram (EEG). Blood data and cerebral blood flow and metabolism were measured throughout and at "insults," i.e., at apnea and cessation events, to clarify their interdependence. Arterial and brain venous PO2 fell linearly with inspired oxygen (final value of 2% at 280 s). Hyperpnea induced arterial alkalosis; subsequent hypopnea led to near-normal PCO2 and pH when EEG ceased. Hypercapnia was more pronounced in cerebral than in systemic venous blood; time courses of pH changes were similar. Sagittal sinus blood pressure and outflow were linearly related and resembled the time course of local cerebral blood flow. Blood flow increased by 25% at apnea and only 60% at EEG silence. Cerebral metabolic rate of O2 rose during the hyperpnea phase and fell exponentially thereafter. Cerebral glucose uptake and lactate release increased within the first 3 min but fell abruptly when cortico-electric spiking ceased. Time courses of cerebral O2 consumption and spike rate were linearly related; both showed inverse linear relations to cerebral perfusion. The hypoxic insults were well defined by blood data; critical PO2 values were lower than previously assumed. This model is proving to be a useful, controlled method by which mechanisms of cerebral hypoxia tolerance may be studied in vivo.     

Identifying populations useful for improving parents of a single cross based on net transfer of alleles

Summary Theory and methods for identifying populations (P _y ) with the highest frequency of favorable dominant alleles not present in an elite single cross (I ₁× I ₂) have been developed recently. During selection, new favorable alleles can be transferred from P _y to either I ₁ or I ₂ only at the risk of losing favorable alleles already present in the single cross. A net improvement (NI) statistic, which estimates the relative number of favorable alleles that can be gained from P _y minus the relative number of favorable alleles that can be lost from I ₁ or I ₂, is presented. NI is calculated as maximum [(I ₁×P _y –I ₁×I ₂)/2,(I ₂×P _y –I ₁×I ₂)/2]. Because I ₁ × I ₂ is constant in an experiment, the method reduces to choosing P _y populations with the best mean performance in combination with either I ₁ or I ₂. For a set of maize (Zea mays L.) grain yield data, NI was highly correlated to three other statistics proposed for choosing populations, namely: (1) minimally biased estimate (l ) of the relative number of favorable dominant alleles present in P _y but not in I ₁ and I ₂; (2) minimum upper bound on l ; and (3) predicted performance of the three-way cross [P _y (I ₁× I ₂)]. While l estimates potential improvement likely to be achieved only through long-term recurrent selection, NI is probably a better predictor of short-term improvement in single-cross performance.A contribution from Lifaco Genetics, a subsidiary of Groupe Limagrain     

Chaetognaths: a useful model for studying heat shock proteins. Effect of wound healing

The pattern of expression of Heat Shock Protein 70 (Hsp70), a highly conserved cellular protein chaperone, was investigated in Chaetognatha, a very important phylum of marine worms, which play a major role in marine food webs. The in toto distribution of Hsp70-like immunoreactivity was assessed in both intact and experimentally wounded specimens of Spadella cephaloptera Busch, 1851 that have been cut transversally just above the seminal vesicles. In intact animals, the ciliary organs, the corona ciliata, the coronal nerve and the spermatocytes express Hsp70 proteins. The kinetic of the expression pattern has been followed during the wound healing from the lesion to 5 days after. The Hsp70 immunoreactivity was observed according to time in the cerebral and suboesophageal ganglia and in the plexuses of the hood, the mouth, the neck and the tail region. At the wound level, the time-dependent Hsp70 expression was detected in the epidermal cells and along numerous muscle fibres of the tail region. Five days after the tail section, two Hsp70 immunoreactive areas were observed on both sides of the healed wound that correspond to the sites of formation of two new seminal vesicles. It is suggested that, in Chaetognaths, Hsp70 may be implicated in the regulation of several cellular processes especially at the level of the spermatocytes in intact and wounded specimens, and of the nervous system and muscular apparatus during the wound healing. It appears that chaetognaths are a good model as indicator of stress responses for experimental studies at the level of a whole organism.     

Development of a magnetic biocatalyst useful for the synthesis of ethyloleate

Candida antarctica Lipase B was successfully immobilized on magnetite (Fe₃O₄) nanoparticles functionalized with chitosan and glutaraldehyde. The obtained magnetic catalyst was characterized and its performance was evaluated in solvent-free synthesis of ethyl oleate at room temperature. The performance of this biocatalyst was compared with the commercial Novozym 435, as a tool to estimate the efficiency of immobilization. It was found that using 33 mg of the biocatalyst it was possible to reach almost the same activity that was obtained using 12 mg of Novozym 435. Furthermore, this new biocatalyst presents the advantages of not being degraded by short alcohols, being easily recovered from the reaction media by magnetic decantation, and low fabrication cost. The possibility of reutilization was also studied, keeping a significant activity up to eight cycles. A special sampling protocol was also developed for the multiphasic reaction system, to assure accurate results. This novel biocatalyst is an interesting alternative for potential industrial applications, considering the above-mentioned advantages.     

Development of a bacterial model for studying anthracycline-membrane interactions

Abstract Growth of wild-type Escherichia coli strain MRE600 was severely affected up to 9 h following treatment with the anthracycline doxorubicin (15 μM), however, after 9 h, the cells became resistant. The onset of resistance coincided with some changes in the relative proportions of total saturated, monounsaturated and cyclopropane fatty acids. The anionic lipid content in E. coli strain HDL11 is under lac control and synthesis can be induced by incubation with the lac inducer IPTG. HDL11, with low levels of anionic phospholipid, was unaffected by doxorubicin (100 μM) over 9 h, with only slight inhibition of growth seen over 24 h. When the anionic lipid content of HDL11 was increased, there was a slight increase in the efficacy of doxorubicin, providing evidence for a membrane-based step in doxorubicin action.