RNA interference and its role in the regulation of eucaryotic gene expression

Several years ago it was discovered that plant transformation with a transcribed sense transgene could shut down the expression of a homologous endogenous gene. Moreover, it was shown that the introduction into the cell of dsRNA (double-stranded RNA) containing nucleotide sequence complementary to an mRNA sequence causes selective degradation of the latter and thus silencing of a specific gene. This phenomenon, called RNA interference (RNAi) was demonstrated to be present in almost all eukaryotic organisms. RNAi is also capable of silencing transposons in germ line cells and fighting RNA virus infection. Enzymes involved in this process exhibit high homology across species. Some of these enzymes are involved in other cellular processes, for instance developmental timing, suggesting strong interconnections between RNAi and other metabolic pathways. RNAi is probably an ancient mechanism that evolved to protect eukaryotic cells against invasive forms of nucleic acids.     

Confirmation of Escherichia coli and its distinction from Klebsiella species by gas and indole formation at 44 and 44.5 degrees C

AIMS: In the enumeration of coliform bacteria, confirmation of Escherichia coli has been based upon gas and indole production at the elevated incubation temperature. The test for gas production has recently been questioned. The aim of this study was to investigate the impact of gas production test on the reliability of confirmation of E. coli. METHODS AND RESULTS: The impact of several media on growth, gas and/or indole formation was tested at 44 and 44.5 degrees C using 547 environmental isolates. These were mainly E. coli, Klebsiella pneumoniae, K. oxytoca and Enterobacter cloacae strains. Another set of 250 faecal and environmental klebsiellae were tested for their maximum temperature for growth (Tmax) and for gas formation. Escherichia coli and even K. pneumoniae grew well in all the media, but gas production was more dependent on the medium used. Growth of the mainly gas negative Ent. cloacae and K. oxytoca strains was still more sensitive to the medium and incubation conditions. Tryptophan salt broth was the most productive medium for the indole test, followed by lauryl tryptose mannitole and tryptone mannitol ricinoleate broth (TRM). Tmax of K. oxytoca was clearly lower than Tmax of K. pneumoniae but a rather high fraction of its isolates produced indole at 44.5 degrees C. CONCLUSIONS: False-positive E. coli confirmation is possible if gas production is not tested for and the confirmation is based on indole test only. SIGNIFICANCE AND IMPACT OF THE STUDY: Erroneous positive results on routine analysis for E. coli can occur.     

Crystal structure of an in vitro affinity- and specificity-matured anti-testosterone Fab in complex with testosterone. Improved affinity results from small structural changes within the variable domains

A highly selective, high affinity recombinant anti-testosterone Fab fragment has been generated by stepwise optimization of the complementarity-determining regions (CDRs) by random mutagenesis and phage display selection of a monoclonal antibody (3-C(4)F(5)). The best mutant (77 Fab) was obtained by evaluating the additivity effects of different independently selected CDR mutations. The 77 Fab contains 20 mutations and has about 40-fold increased affinity (K(d) = 3 x 10(-10) m) when compared with the wild-type (3-C(4)F(5)) Fab. To obtain structural insight into factors, which are needed to improve binding properties, we have determined the crystal structures of the mutant 77 Fab fragment with (2.15 A) and without testosterone (2.10 A) and compared these with previously determined wild-type structures. The overall testosterone binding of the 77 Fab is similar to that of the wild-type. The improved affinity and specificity of the 77 Fab fragment are due to more comprehensive packing of the testosterone with the protein, which is the result of small structural changes within the variable domains. Only one important binding site residue Glu-95 of the heavy chain CDR3 is mutated to alanine in the 77 Fab fragment. This mutation, originally selected from the phage library based on improved specificity, provides more free space for the testosterone D-ring. The light chain CDR1 of 77 Fab containing eight mutations has the most significant effect on the improved affinity, although it has no direct contact with the testosterone. The mutations of CDR-L1 cause a rearrangement in its conformation, leading to an overall fine reshaping of the binding site.     

Characterization of a novel killer cell lectin-like receptor (KLRH1) expressed by alloreactive rat NK cells

NK cells have the ability to recognize and kill MHC-mismatched hemopoietic cells. In the present study, strain-specific differences in the rat NK allorecognition repertoire were exploited to generate Abs against receptors that may be involved in allogeneic responses. A mAb termed STOK9 was selected, and it reacted with subsets of NK cells and NKR-P1(+) T cells from certain rat strains possessing highly alloreactive NK cells. The STOK9(+) NK subset was broadly alloreactive and lysed Con A lymphoblast targets from a range of MHC-mismatched strains. The mAb STOK9 precipitated a 75-kDa dimeric glycoprotein from NK lysates. Expression cloning revealed that each monomer consisted of 231 aa with limited homology to other previously characterized killer cell lectin-like receptors (KLRs). This glycoprotein therefore constitutes a novel KLR branch, and it has been termed KLRH1. A gene in the central region of the natural killer gene complex on rat chromosome 4 encodes KLRH1. A mouse homolog appears to be present as deduced from analyses of genomic trace sequences. The function of KLRH1 is unknown, but it contains an immunoreceptor tyrosine-based inhibitory motif, suggesting an inhibitory function. The MHC haplotype of the host appears to influence KLRH1 expression, suggesting that it may function as an MHC-binding receptor on subsets of NK cells and T lymphocytes.     

The establishment of a network of European human research tissue banks

This is a report of a workshop held on the establishment of human research tissue banking which was held in Levi, Finland 21–24 March 2002.There were 21 participants from 7 European countries. This meeting was attended by representatives from academia, research tissue banks and from the Biotech and Pharmaceutical Industries. The principal aim of the workshop was to find a way to progress the recommendations from ECVAM workshop 44 (ATLA 29, 125–134,2001) and ECVAM workshop 32 (ATLA 26, 763–777, 1998). The workshop represented the first unofficial meeting of the European Network of Research Tissue Banks (ENRTB) steering group. It is expected that in the period preceding the next workshop the ENRTB steering group will co-ordinate the ethical,legislative and organisational aspects of research tissue banking. Key issues dealt with by the Levi workshop included the practical aspects of sharing expertise and experiences across the different European members. Such collaboration between research tissue banks and end users of such material seeks to ultimately enable shared access to human tissue for medical and pharmaco-toxicological research while maintaining strict adherence to differences in legal and ethical aspects related to the use of human tissue in individual countries. This revised version was published online in July 2006 with corrections to the Cover Date.     

Essential oil composition in leaves of carrot varieties and preference of specialist and generalist sucking insect herbivores

• 1 The carrot psyllid Trioza apicalis and Lygus rugulipennis can cause serious damage to cultivated carrots, especially at the seedling stage. The essential oil composition in leaves of several carrot varieties was studied in relation to oviposition acceptance by sucking insect herbivores that have different feeding strategies. Of the insect species studied, T. apicalis is a carrot specialist, T. anthrisci is an Apiaceae specialist, and L. rugulipennis is a generalist.
• 2 The oil composition differed significantly between leaves from different carrot varieties (Flakkeer 2, Nantura, Parano, Napoli, Panther, Splendid, Nantes 3 Express).
• 3 There were no differences in the mean numbers of eggs laid by T. apicalis or T. anthrisci on different varieties, but L. rugulipennis laid fewer eggs on the Panther variety than on Nantes 3 Express.
• 4 There was a negative linear correlation between limonene concentration and number of eggs laid by T. apicalis, confirming that limonene is a repellent to the carrot psyllid. Results suggest that only particular compounds influence the preference of the carrot psyllid, and these compounds may not be the main components in the essential oil of carrots.
• 5 A positive correlation was found between myrcene concentration and number of eggs laid by T. anthrisci. Essential oil composition did not explain egg‐laying preference of L. rugulipennis.
• 6 The results indicate that essential oil composition of carrot varieties affects host preference of Apiaceae specialists more than that of generalists.

     

Presumptive fecal streptococci in environmental samples characterized by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

The use of fecal streptococci as fecal indicators requires better knowledge of the ecology of these bacteria. We isolated 371 presumptive fecal streptococci from environmental samples--domestic wastewater, forest industry wastewater, contaminated surface and seawater, well water, cow dung, bird droppings, and pristine waters--and clustered them according to their protein profiles in one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Some clusters could be tentatively identified with the help of reference strains. Samples from each environment had a typical composition of streptococcus types. Enterococcus faecalis was present, but not as a dominating enterococcal species, in samples in which fecal contamination was probable. Enterococcus faecium, Enterococcus durans, Enterococcus hirae, and Enterococcus mundtii had protein profiles that were difficult to distinguish from each other. These bacteria were found in a variety of samples. Enterococcus casseliflavus and Enterococcus gallinarum had identical protein profiles. On the basis of the maximum temperatures for growth and pigment production, isolates of this protein profile group common in forest industry wastewaters were identified as E. casseliflavus. Lactococcus lactis subsp. lactis was also found in this environment. Nearly all strains from pristine waters belonged to protein profile groups which could not be identified with the aid of known Aerococcus, Enterococcus, Lactococcus, or Streptococcus strains. The maximum temperatures for growth and the results of fatty acid analysis were in general agreement within each protein profile group.     

Dopamine Stimulates K+ Efflux in the Chick Retina via D1 Receptors Independently of Adenylyl Cyclase Activation

Abstract: Dopamine (DA) stimulated K⁺ efflux (assessed as ⁸⁶Rb⁺ efflux) in retinal suspensions of posthatched chicken. This effect was dose dependent (EC₅₀= 22 μM), was mimicked by the D₁-selective antagonist SKF-38393, and reversed by the D₁-selective antagonist SCH-23390, indicating an involvement of D₁ receptors. Analogues of cyclic AMP (CAMP) did not mimic the DA action. Moreover, DA failed to affect cAMP levels, suggesting that adenylyl cyclase (AC) was not involved. In contrast, forskolin (FSK) stimulated both K⁺ efflux and cAMP accumulation in the retina (EC₅₀ of 10 μM for both effects). The FSK-elicited K⁺ efflux was not mimicked by 1,9-dideoxy-FSK (an analogue of FSK that does not activate AC), suggesting that FSK stimulated K⁺ efflux through the activation of AC. Both DA and FSK inhibited Na⁺,K⁺-ATPase activity in the retina. However, the DA-elicited K* efflux was independent of this inhibition, whereas the FSK effect on K⁺ efflux was largely due to the inhibitory action of the diterpene of the ion pump. A possible role of protein kinase C (PKC) in the DA action was explored. The PKC activator 4β-phorbol 12-myristate 13-acetate (4β-PMA) potently (EC₅₀= 4 nM) stimulated K⁺ efflux. This action was not mimicked by the inactive isomer 4α-PMA. When added together, DA and 4β-PMA behaved in an additive manner, suggesting separate mechanisms of action for these two drugs. Moreover, DA failed to stimulate retinal phosphoinositide hydrolysis, a well-known pathway leading to PKC activation. These data suggest that DA acting through D₁ receptors and independently of AC can modulate its target cell excitability in the chick retina by stimulating K⁺ efflux pathways. The mechanism of the DA action remains to be clarified.     

Antibiotic resistance among different species of fecal coliforms isolated from water samples.

The distribution of resistance to ampicillin, chloramphenicol, sulfonamides, tetracycline, and streptomycin among fecal coliforms in sewage, surface waters, and sea water was investigated. The incidence of resistant strains among isolates varied significantly among the water samples, without obvious connection with the water source or the level of pollution. The average frequency of multiple resistance was not always high in the same samples in which the overall resistance was high. The species composition varied considerably in different water samples. A significant correlation was observed between the relative frequency of Klebsiella species and the incidence of ampicillin resistance in water samples. The importance of species composition of fecal coliforms, affected by their source and by the aquatic environment, on the resistance pattern is noted.