Mutational study of the bacterial hemoglobin distal heme pocket

Ligand binding experiments on three mutants in the distal heme pocket of Vitreoscilla hemoglobin (GlnE7His, ProE8Ala, and GlnE7His,ProE8Ala) were used to probe the role of GlnE7 and ProE8 in the pocket's unusual structure. The oxygen dissociation constants for the wild type, E8Ala mutant, and E7His mutant proteins were 4.5, 4.7, and 1.7microM, respectively; the K(d) for the double mutant was not determinable by our technique. Visible-Soret spectra of the carbonyl and cyanyl forms and FT-IR of the carbonyl form of the E8 mutant were similar to those of the wild type; the opposite was true for the GlnE7His and GlnE7His,ProE8Ala mutants, which also differed from wild type in the visible-Soret spectra of their oxidized forms. Models of the effects of the mutations on distal pocket structure were consistent with the experimental findings, particularly the larger effects of the GlnE7His change.     

The influence of water on the nanomechanical behavior of the plant biopolyester cutin as studied by AFM and solid-state NMR

Atomic force microscopy and solid-state nuclear magnetic resonance have been used to investigate the effect of water absorption on the nanoscale elastic properties of the biopolyester, cutin, isolated from tomato fruit cuticle. Changes in the humidity and temperature at which fruits are grown or stored can affect the plant surface (cuticle) and modify its susceptibility to pathogenic attack by altering the cuticle's rheological properties. In this work, atomic force microscopy measurements of the surface mechanical properties of isolated plant cutin have been made as a first step to probing the impact of water uptake from the environment on surface flexibility. A dramatic decrease in surface elastic modulus (from approximately 32 to approximately 6 MPa) accompanies increases in water content as small as 2 wt %. Complementary solid-state nuclear magnetic resonance measurements reveal enhanced local mobility of the acyl chain segments with increasing water content, even at molecular sites remote from the covalent cross-links that are likely to play a crucial role in cutin's elastic properties.     

Secretion of β-lactamase from K. lactis using pEPS1, a convenient episomal vector designed for the secretion of foreign proteins

Summary A convenient shuttle vector that enables high level secretion of proteins from Kluyveromyces lactis has been developed. The vector, pEPS1, contains a unique cloning site that allows the construction, in a single ligation step, of episomal plasmids capable of directing secretion of foreign gene products from K. lactis. As an example we demonstrate the production of -lactamase and determine optimal conditions for its secretion into the culture media.     

Physiological history may mask the inherent inducible desiccation tolerance strategy of the desert moss Crossidium crassinerve

Shoots of bryophytes collected in the desiccated state from the field are likely to be hardened to desiccation tolerance (DT) to varying degrees. To account for this, most studies on DT include a relatively short deacclimation period. However, no study has experimentally determined the appropriate deacclimation time for any bryophyte species. Our purposes are to (i) determine if ‘field effects’ are biologically relevant to DT studies and how long a deacclimation period is required to remove them; and (ii) utilise field versus cultured shoot responses within the context of a deacclimation period to elucidate the ecological strategy of DT. Our hypothesis (based on an extensive literature on DT) is that a deacclimation period from 24 to 72 h should be sufficient to eliminate historical stress effects on the physiology of the shoots and allow an accurate determination of the inherent ecological DT strategy (constitutive or inducible). We determined, however, using chlorophyll fluorescence and visual estimates of shoot damage, that field‐collected shoots of the desert moss Crossidium crassinerve required an experimental deacclimation period of >7 days before field effects were removed, and revealed an ecological DT strategy of inducible DT. If the deacclimation period was <6 days, the shoot response conformed to an ecological strategy of constitutive protection. Thus the presence of field effects can obscure the ecological strategy of desiccation tolerance exhibited by the species, and this translates into a need to re‐evaluate previous mechanistic and ecological studies of desiccation tolerance in plants.     

Evidence that Na+-pumping occurs through the D-channel in Vitreoscilla cytochrome bo

The operon (cyo) encoding the Na(+)-pumping respiratory terminal oxidase (cytochrome bo) of the bacterium Vitreoscilla was transformed into Escherichia coli GV100, a deletion mutant of cytochrome bo. This was done for the wild type operon and five mutants in three conserved Cyo subunit I amino acids known to be crucial for H(+) transport in the E. coli enzyme, one near the nuclear center, one in the K-channel, and one in the D-channel. CO-binding, NADH and ubiquinol oxidase, and Na(+)-pumping activities were all substantially inhibited by each mutation. The wild type Vitreoscilla cytochrome bo can pump Na(+) against a concentration gradient, resulting in a transmembrane concentration differential of 2-3 orders of magnitude. It is proposed that Vitreoscilla cytochrome bo pumps four Na(+) through the D-channel to the exterior and transports four H(+) through the K-channel for the reduction of each O(2).     

The bacterial paromomycin resistance gene, aphH, as a dominant selectable marker in Volvox carteri

The aminoglycoside antibiotic paromomycin that is highly toxic to the green alga Volvox carteri is efficiently inactivated by aminoglycoside 3′-phosphotransferase from Streptomyces rimosus. Therefore, we made constructs in which the bacterial aphH gene encoding this enzyme was combined with Volvox cis-regulatory elements in an attempt to develop a new dominant selectable marker – paromomycin resistance (Pm^R) – for use in Volvox nuclear transformation. The construct that provided the most efficient transformation was one in which aphH was placed between a chimeric promoter that was generated by fusing the Volvox hsp70 and rbcS3 promoters and the 3′ UTR of the Volvox rbcS3 gene. When this plasmid was used in combination with a high-impact biolistic device, the frequency of stable Pm^R transformants ranged about 15 per 10⁶ target cells. Due to rapid and sharp selection, Pm^R transformants were readily isolated after six days, which is half the time required for previously used markers. Co-transformation of an unselected marker ranged about 30%. The chimeric aphH gene was stably integrated into the Volvox genome, frequently as tandem multiple copies, and was expressed at a level that made selection of Pm^R transformants simple and unambiguous. This makes the engineered bacterial aphH gene an efficient dominant selection marker for the transformation and co-transformation of a broad range of V. carteri strains without the recurring need for using auxotrophic recipient strains.     

Large-scale recording of neurons by movable silicon probes in behaving rodents

A major challenge in neuroscience is linking behavior to the collective activity of neural assemblies. Understanding of input-output relationships of neurons and circuits requires methods with the spatial selectivity and temporal resolution appropriate for mechanistic analysis of neural ensembles in the behaving animal, i.e. recording of representatively large samples of isolated single neurons. Ensemble monitoring of neuronal activity has progressed remarkably in the past decade in both small and large-brained animals, including human subjects. Multiple-site recording with silicon-based devices are particularly effective because of their scalability, small volume and geometric design. Here, we describe methods for recording multiple single neurons and local field potential in behaving rodents, using commercially available micro-machined silicon probes with custom-made accessory components. There are two basic options for interfacing silicon probes to preamplifiers: printed circuit boards and flexible cables. Probe supplying companies (http://www.neuronexustech.com/; http://www.sbmicrosystems.com/; http://www.acreo.se/) usually provide the bonding service and deliver probes bonded to printed circuit boards or flexible cables. Here, we describe the implantation of a 4-shank, 32-site probe attached to flexible polyimide cable, and mounted on a movable microdrive. Each step of the probe preparation, microdrive construction and surgery is illustrated so that the end user can easily replicate the process.     

The relative strengths of SR protein-mediated associations of alternative and constitutive exons can influence alternative splicing.

We have characterized the functional role of SR protein-mediated exon/exon associations in the alternative splicing of exon 5 of chicken cardiac troponin T (cTnT). We have previously shown that SR proteins can promote the association of the alternative exon 5 with the flanking constitutive exon 6 of this pre-mRNA. In this study, we have shown that when exons 2, 3, and 4 of the cTnT pre-mRNA are spliced together, the composite exon 2/3/4 contains an additional SR protein binding site. Furthermore, we have found that SR proteins can also promote interactions between the pairs of exons 2/3/4-5 and 2/3/4-6. We then asked whether the SR protein binding sites in these exons play a role in cTnT alternative splicing in vivo. We found that the SR protein binding sites in exons 2/3/4 and 6 promote exon 5 skipping, and it has previously been shown that the SR protein binding site in exon 5 promotes exon 5 inclusion. Consistent with these results, we find that the SR protein-mediated association of exon 2/3/4 with 6 is preferred over associations involving exon 5, in that exons 2/3/4 and 6 are more efficient than exon 5 in competing an SR protein-mediated exon/exon association. We suggest that the relative strengths of SR protein-mediated associations of alternative and constitutive exons play a role in determining alternative splicing patterns.     

Enzymatic synthesis of tea theaflavin derivatives and their anti-inflammatory and cytotoxic activities

Derivatives based on a benzotropolone skeleton (9-26) have been prepared by the enzymatic coupling (horseradish peroxidase/H2O2) of selected pairs of compounds (1-8), one with a vic-trihydroxyphenyl moiety, and the other with an ortho-dihydroxyphenyl structure. Some of these compounds have been found to inhibit TPA-induced mice ear edema, nitric oxide (NO) synthesis, and arachidonic acid release by LPS-stimulated RAW 264.7 cells. Their cytotoxic activities against KYSE 150 and 510 human esophageal squamous cell carcinoma and HT 29 human colon cancer cells were also evaluated.