Serologic and immunochromatographic detection of oxygenated polyenoic acids in Euglena gracilis var. bacillaris

The extent of evolutionary conservation of DNA complimentary to RNA stored in the mature oocyte of the sea urchin S. purpuratus has been assessed. To do this, such DNA was hybridized with total genomic DNA of S. purpuratus and S. franciscanus and the thermal stability of the resultant duplexes was measured by two methods. In the first method, the duplexes were bound to hydroxylapatite and thermally eluted; the difference in thermal stability between homologous and heterologous duplexes averaged 6.9 degrees C in duplicate determinations. In the second experiment, the same hybrids were thermally melted in 2.4M tetraethylammonium chloride, then assayed with S1 nuclease; the difference in thermal stability of homologous and heterologous duplexes was 4.8 degrees C. Either value is significantly lower than the divergence of total single-copy DNA among these species as measured by the same techniques. This demonstrates that DNA sequences complimentary to maternal RNA are conserved during evolution, and thus that a high fraction of them are likely to be physiologically functional.     

Light-independent and dependent phases of proplastid development in Euglena gracilis W3BUL

Cells of Euglena gracilis Klebs var. bacillaris Cori mutant W3BUL grown in darkness on Hutner's pH 3.5 medium without agitation accumulate wax ester. These cells have undeveloped proplastid remnants characteristic of this mutant. If these cells are transferred to an inorganic medium and bubbled with 2-3% CO2 in air, the wax disappears and the proplastid expands and develops in darkness to form prolamellar bodies and membrane vessicles within 96 h. No further development takes place in darkness, but if these cultures are illuminated at 96 h formation of prothylakoids is observed. Thus the wax ester accumulated during growth can be used subsequently to support proplastid development up to the prolamellar body stage, but the formation of prothylakoids is strictly light-dependent. Development in this system takes place at a slower rate than in cells grown with shaking and lacking wax which are transferred to resting medium. As previously shown, all of proplastid development requires light under these conditions. It is suggested that the oxygen-requiring utilization of wax in darkness can provide energy and metabolites for a part of proplastid development but the later steps in these cells, or the entire development in cells lacking wax is supported by paramylum degradation which is strictly light-dependent. However, a specific light reaction required for prothylakoid organization is not ruled out.     

Ovine pedomics: the first study of the ovine foot 16S rRNA-based microbiome

We report the first study of the bacterial microbiome of ovine interdigital skin based on 16S rRNA by pyrosequencing and conventional cloning with Sanger-sequencing. Three flocks were selected, one a flock with no signs of footrot or interdigital dermatitis, a second flock with interdigital dermatitis alone and a third flock with both interdigital dermatitis and footrot. The sheep were classified as having either healthy interdigital skin (H) and interdigital dermatitis (ID) or virulent footrot (VFR). The ovine interdigital skin bacterial community varied significantly by flock and clinical condition. The diversity and richness of operational taxonomic units was greater in tissue from sheep with ID than H or VFR-affected sheep. Actinobacteria, Bacteriodetes, Firmicutes and Proteobacteria were the most abundant phyla comprising 25 genera. Peptostreptococcus, Corynebacterium and Staphylococcus were associated with H, ID and VFR, respectively. Sequences of Dichelobacter nodosus, the causal agent of ovine footrot, were not amplified because of mismatches in the 16S rRNA universal forward primer (27F). A specific real-time PCR assay was used to demonstrate the presence of D. nodosus, which was detected in all samples including the flock with no signs of ID or VFR. Sheep with ID had significantly higher numbers of D. nodosus (10⁴–10⁹ cells per g tissue) than those with H or VFR feet.     

Mammalian reoviruses contain a myristoylated structural protein.

The structural protein mu 1 of mammalian reoviruses was noted to have a potential N-myristoylation sequence at the amino terminus of its deduced amino acid sequence. Virions labeled with [3H]myristic acid were used to demonstrate that mu 1 is modified by an amide-linked myristoyl group. A myristoylated peptide having a relative molecular weight (Mr) of approximately 4,000 was also shown to be a structural component of virions and was concluded to represent the 4.2-kDa amino-terminal fragment of mu 1 which is generated by the same proteolytic cleavage that yields the carboxy-terminal fragment and major outer capsid protein mu 1C. The myristoylated 4,000-Mr peptide was found to be present in reovirus intermediate subviral particles but to be absent from cores, indicating that it is a component of the outer capsid. A distinct large myristoylated fragment of the intact mu 1 protein was also identified in intermediate subviral particles, but no myristoylated mu-region proteins were identified in cores, consistent with the location of mu 1 in the outer capsid. Similarities between amino-terminal regions of the reovirus mu 1 protein and the poliovirus capsid polyprotein were noted. By analogy with other viruses that contain N-myristoylated structural proteins (particularly picornaviruses), we suggest that the myristoyl group attached to mu 1 and its amino-terminal fragments has an essential role in the assembly and structure of the reovirus outer capsid and in the process of reovirus entry into cells.     

The dissociation between perception and action in the Ebbinghaus illusion: nonillusory effects of pictorial cues on grasp

According to a recently proposed distinction [1] between vision for perception and vision for action, visually guided movements should be largely immune to the perceptually compelling changes in size produced by pictorial illusions. Tests of this prediction that use the Ebbinghaus illusion have revealed only small effects of the illusion on grasp scaling as compared to its effect on perception [2-4]. Nevertheless, some have argued that the small effect on grasp implies that there is a single representation of size for both perception and action [5]. Recent findings, however, suggest that the 2-D pictorial elements, such as those comprising illusory backgrounds, can sometimes be treated as obstacles and thereby influence the programming of grasp [6]. The arrangement of the 2-D elements commonly used in previous studies examining the Ebbinghaus illusion could therefore give rise to an effect on grasp scaling that is independent of its effect on perceptual judgements, even though the two effects are in the same direction. We present evidence demonstrating that when the gap between the target and the illusion-making elements in the Ebbinghaus illusion is equidistant across different perceptual conditions (Figure 1a), the apparent effect of the illusion on grasp scaling is eliminated.     

Efficient, chemoselective synthesis of immunomicelles using single-domain antibodies with a C-terminal thioester

Background  

Classical bioconjugation strategies for generating antibody-functionalized nanoparticles are non-specific and typically result in heterogeneous compounds that can be compromised in activity. Expression systems based on self-cleavable intein domains allow the generation of recombinant proteins with a C-terminal thioester, providing a unique handle for site-specific conjugation using native chemical ligation (NCL). However, current methods to generate antibody fragments with C-terminal thioesters require cumbersome refolding procedures, effectively preventing application of NCL for antibody-mediated targeting and molecular imaging.