10,20-Methanorhodopsins: (7E,9E,13E)-10,20-methanorhodopsin and (7E,9Z,13Z)-10,20-methanorhodopsin. 11-cis-locked rhodopsin analog pigments with unusual thermal and photo-stability

Synthesis of the retinal analog, 10,20-methanoretinal (R6), where the 11Z conformation is locked in a six-membered ring, yielded four stereoisomers (7E,9E,13E, 7E,9E,13Z, 7E,9Z,13E and 7E,9Z,13Z). These four isomers were separated by straight-phase isocratic HPLC and identified by 1H-NMR and NOE analysis. All isomers smoothly recombined with bovine opsin at a relatively high rate (5-10% of that of the natural chromophore 11Z-retinal). The corresponding 13E and 13Z isomers yielded identical analog pigments, probably due to rapid thermal isomerization around the C13 = C14 double bond. The (7E,9E)-isomers produced a pigment with maximal absorbance at 510 nm, while the pigment produced from the (7E,9Z)-isomers had maximal absorbance at 494 nm. Based upon kinetic considerations, the chromophore structure in the 510-nm-absorbing pigment should be (7E,9E,13E), i.e. equivalent to 11Z-retinal and rhodopsin, while the chromophore structure in the 494-nm-absorbing pigment should be (7E,9Z,13Z), i.e. equivalent to (9Z,11Z,13Z)-rhodopsin, an isorhodopsin analog. In analogy to the 11-cis-locked rhodopsin analogs Rh5 and Rh7, the 510-nm-absorbing pigment, (7E,9E,13E)-10,20-methanorhodopsin, was dubbed Rh6 and the 494-nm-absorbing pigment. (7E,9Z,13Z)-10,20-methanorhodopsin, was dubbed Iso6. The opsin shift of Rh6 (2660 cm-1) is practically identical to that of rhodopsin itself (2650 cm-1). Rh6 and Iso6 are nearly as stable as rhodopsin towards hydroxylamine and solubilization in detergent solution and could be easily purified and reconstituted into proteoliposomes by established procedures. Due to the 11-cis-lock, Rh6 is much less photolabile (bleaching rate less than 1%) than rhodopsin, but it is not completely photostable, probably since photoisomerization around the C7 = C8, C9 = C10 and C13 = C14 bonds is allowed. Illumination of either Rh6 or Iso6 does not generate the common photointermediates but instead produces a complex pattern of photochemical transitions, which during continuous illumination leads to the same final steady state, absorbing at 498 nm. This process is accompanied by a slow but steady loss of pigment, probably due to hydrolytic release of chromophore, which is markedly accelerated in the presence of hydroxylamine. In a physiological assay (light-dependent activation of rod cGMP phosphodiesterase) Rh6 is only marginally active and this probably reflects conformational changes accompanying the above-mentioned photochemical transitions. This supports the concept that normal rhodopsin-based phototransduction requires 11Z to all-E isomerization.(ABSTRACT TRUNCATED AT 400 WORDS)     

A Method for Histological Preparation of Undecalcified Bone Sections Containing Acrylic Bone Cement

An improved and time reducing method is presented for the histological evaluation of bone containing polymethylmethacrylate (PMMA) bone cement. The undecalcified bone was embedded in epoxy resin and sections of 50-100 μm thickness were produced using a commercially available cutting grinding system. The sections were stained with Stevenel's blue and van Gieson picrofuchsin or a modified hematoxylin-eosin. PMMA bone cement was not dissolved and remained enabling examination in situ of an intact cement bone interface and tissue reaction without decalcification.     

Methodological problems in evolutionary biology

One of the major criticisms of optimal foraging theory (OFT) is that it is not testable. In discussions of this criticism opposing parties have confused methodological concepts and used meaningless biological concepts. In this paper we discuss such misunderstandings and show that OFr has an empirically testable, and even well-confirmed, general core theory. One of our main conclusions is that specific model testing should not be aimed at proving optimality, but rather at identifying the context in which certain types of behaviour are optimal. To do this, it is necessary to be aware of the assumptions made in testing a model. The assumptions that are explicitly stated in the literature up to now do not completely cover the actual assumptions made in testing OFT models in practice. We present a more comprehensive set of assumptions. Although all the assumptions play a role in testing models, they are not of equal status. Crucial assumptions concern constraints and the relation between fitness and currency. Therefore, it is essential to make such assumptions testable in practice. We show that a more explicit relationship between OFT modelling and evolutionary theory can help with this. Specifically, phylogeny reconstruction and population dynamic modelling can and should be used to formulate assumptions concerning constraints and currencies.     

Alteration in molecular structure which results in disease: the Met-30 variant of human plasma transthyretin.

The structure of a variant transthyretin has been determined by X-ray crystallography at 2.3 A resolution in order to investigate those changes which lead to amyloid formation. This variant transthyretin, in which the internal valyl residue at position 30 is replaced by methionyl, is associated with the most common form of familial amyloidotic polyneuropathy (FAP). Comparison to the known structure of the normal transthyretin tetramer shows that the bulkier methionine residue 30 which lies between the nearly orthogonal beta sheets of the dimer, results in the sheets being displaced an average of 0.4 A. The internal structure of the sheets and of the monomer-monomer interface is maintained. Such global changes may affect the metabolic properties and the tendency towards polymerization of the mutant protein. These findings may form a basis for understanding other amyloid-deposition diseases.     

Structure and function of root-inducing (Ri) plasmids and their relation to tumor-inducing (Ti) plasmids

The abilities of Agrobacterium tumefaciens and A. rhizogenes to transform dicotyle-dons and cause crown gall and hairy root disease are caused by the presence of tumor inducing (Ti) and root inducing (Ri) plasmids. During transformation plasmid T-DNA (transferred DNA) is inserted into the plant genome. The T-region is flanked by 25 bp direct repeats, which are essential for transfer. The T-regions contain oncogenes that are expressed in the plants. Some of these code for enzymes that synthesize auxin or cytokinin. Another type, present in Ri plasmids only, appears to impose a high hormone sensitivity on the infected tissue. The T-DNA also contains genes for enzymes synthesizing opines, which the bacteria catabolize. The T-DNA transfer is initiated by the induction of genes in the virulence (vir) region of the plasmid by phenolic compounds secreted by wounded tissue. The products of the vir -genes and of chromosomal genes mediate transfer of T-DNA to the plant cells. Crown gall disease is caused by production of auxin and cytokinin by the transferred T-DNA. The T-DNA of Ri plasmids codes for at least three genes that each can induce root formation, and that together cause hairy root formation from plant tissue. Current results indicate that the products of these genes induce a potential for increased auxin sensitivity that is expressed when the transformed cells are subjected to a certain level of auxin. After this stage the transformed roots can be grown in culture without exogenous supply of hormones.     

Metabolic engineering of Saccharomyces cerevisiae for the production of n-butanol

Background

The thermotolerant methylotrophic yeast Hansenula polymorpha is capable of alcoholic fermentation of xylose at elevated temperatures (45 – 48°C). Such property of this yeast defines it as a good candidate for the development of an efficient process for simultaneous saccharification and fermentation. However, to be economically viable, the main characteristics of xylose fermentation of H. polymorpha have to be improved.

Results

Site-specific mutagenesis of H. polymorpha XYL1 gene encoding xylose reductase was carried out to decrease affinity of this enzyme toward NADPH. The modified version of XYL1 gene under control of the strong constitutive HpGAP promoter was overexpressed on a Δxyl1 background. This resulted in significant increase in the K_M for NADPH in the mutated xylose reductase (K341 → R N343 → D), while K_M for NADH remained nearly unchanged. The recombinant H. polymorpha strain overexpressing the mutated enzyme together with native xylitol dehydrogenase and xylulokinase on Δxyl1 background was constructed. Xylose consumption, ethanol and xylitol production by the constructed strain were determined for high-temperature xylose fermentation at 48°C. A significant increase in ethanol productivity (up to 7.3 times) was shown in this recombinant strain as compared with the wild type strain. Moreover, the xylitol production by the recombinant strain was reduced considerably to 0.9 mg × (L × h)^-1 as compared to 4.2 mg × (L × h)^-1 for the wild type strain.

Conclusion

Recombinant strains of H. polymorpha engineered for improved xylose utilization are described in the present work. These strains show a significant increase in ethanol productivity with simultaneous reduction in the production of xylitol during high-temperature xylose fermentation.     

SALAMI (Spectrum ALignments using high Accuracy Mass and hIgh sensitivity data): how to make the best out of hybrid MS/MS data

We present a software algorithm that combines ion trap and orbitrap product ion spectra acquired in parallel. The hybrid product ion spectra identify more peptides than when using two separate searches for the orbitrap and LTQ data. The program extracts the high-accuracy mass data from the Orbitrap mass analyzer and combines it with the high-sensitivity data analyzed in the LTQ linear ion trap; the m/z values of the high-confidence fragment ions are corrected to orbitrap mass accuracies and the fragment ion intensities are amplified. This approach utilizes the parallel spectrum measurement capabilities of the LTQ-Orbitrap. We present our approach to handling this type of hybrid data, explain our alignment program, and discuss the advantages of the chosen methodology.     

Non-linear mixed models for repeated data assessment of time and temperature effects on conidia production in the fungus Valdensinia heterodoxa

Valdensinia heterodoxaPeyr. has potential for application as abiological control agent of salal (Gaultheria shallon Pursh) in regeneratingforests in the Pacific Northwest region ofNorth America. Conidia production of twoisolates of Valdensinia heterodoxa wasexamined at four temperature levels during a12-day period. Conidia production was stronglyinfluenced by both temperature (with an optimumbetween 16 and 19 °C) and time (peakedat day 9). Replicate (five) variance was highlyheterogeneous and made a conventional approachto the statistical analyses unsuccessful.However, newly developed generalized nonlinearmodels succeeded in identifying significantdifferences between isolates in theirtemperature specificity. One model was based ona time-dependent Poisson process (logisticmodel) modified by an independent nonlineartemperature function (a scaled gamma function).The other was a nonlinear growth model withfixed (time, isolate) and random (replicate)effects. We believe that nonlinear mixed modelsoffer an attractive framework for analysis oflongitudinal count data common in growth andreproduction studies.     

HIV risk behaviors among outpatients with severe mental illness in Rio de Janeiro,Brazil

We conducted the first study to examine rates of sexual activity, sexual risk behaviors, sexual protective behaviors, injection drug use (IDU), needle sharing, and knowledge about HIV/AIDS among outpatients with severe mental illness (SMI) in Rio de Janeiro, Brazil. Using a measure with demonstrated reliability, we found that 42% of 98 patients engaged in vaginal or anal sex within the past three months. Comorbid substance use disorder was significantly associated with sexual activity. Only 22% of sexually active patients used condoms consistently, despite having better HIV knowledge than those who were sexually abstinent. Overall, 45% of patients reported not engaging in any HIV protective behaviors. There were no reports of drug injection. Adults with SMI in Brazil are in need of efficacious HIV prevention programs and policies that can sustain these programs within mental health treatment settings.