Synthesis and Properties of Bacteriorhodopsin Analogs Containing Electron-Density Labels in the Chromophore Moiety

A method for synthesis of retinal analogs labeled with electron-density groups is suggested. The interaction of these polyene compounds with bacterioopsin in apomembrane of Halobacterium salinarum was tested. A retinal analog containing a crown-ether receptor group is able to interact readily with bacterioopsin giving rise to rapid formation of a pigment with absorption maximum at 460 nm. This pigment is capable of undergoing cyclic photoconversion. The crown-bacteriorhodopsin photocycle is extremely slow and its quantum efficiency is very low (3% of that in native bacteriorhodopsin). This photocycle includes an M-like intermediate with a differential absorption maximum at 380 nm. A retinal analog in which the -ionone ring is replaced by ferrocene moiety forms a stable chromoprotein with the main absorption band at 483 nm and a shoulder near 590-610 nm.     

Photoinduced transformation of 14-F-bacteriorhodopsin gelatin films based on both wild type and D96N mutant

Spectral and kinetic transformations were studied in gelatin films made with 14-F wild type (WT) bacteriorhodopsin (BR) and 14-F D96N mutant BR. Unlike the recent study of water suspensions of the same pigments, where a red shifted species at 660 nm was shown to form under the light in 14-F WT only, there are no drastic differences in photoinduced behavior between gelatin films based on 14-F WT and 14-F D96N. It is not observed any photoinduced formation of red shifted species at 660 nm for both types of films as it is observed for corresponding pigments in water suspension. The observed results are explained in a terms of relationship between the rates of two photoinduced processes that occur in suspensions and films of corresponding pigments. Kinetic characteristics of the photoinduced processes for the films with chemical additives suggest that there are no advantages in using 14-F D96N films when compared to films based on 14-F WT.     

Crystallographic structures of the M and N intermediates of bacteriorhodopsin: assembly of a hydrogen-bonded chain of water molecules between Asp-96 and the retinal Schiff base

An M intermediate of wild-type bacteriorhodopsin and an N intermediate of the V49A mutant were accumulated in photostationary states at pH 5.6 and 295 K, and their crystal structures determined to 1.52A and 1.62A resolution, respectively. They appear to be M(1) and N' in the sequence, M(1)<-->M(2)<-->M'(2)<-->N<-->N'-->O-->BR, where M(1), M(2), and M'(2) contain an unprotonated retinal Schiff base before and after a reorientation switch and after proton release to the extracellular surface, while N and N' contain a reprotonated Schiff base, before and after reprotonation of Asp96 from the cytoplasmic surface. In M(1), we detect a cluster of three hydrogen-bonded water molecules at Asp96, not present in the BR state. In M(2), whose structure we reported earlier, one of these water molecules intercalates between Asp96 and Thr46. In N', the cluster is transformed into a single-file hydrogen-bonded chain of four water molecules that connects Asp96 to the Schiff base. We find a network of three water molecules near residue 219 in the crystal structure of the non-illuminated F219L mutant, where the residue replacement creates a cavity. This suggests that the hydration of the cytoplasmic region we observe in N' might have occurred spontaneously, beginning at an existing water molecule as nucleus, in the cavities from residue rearrangements in the photocycle.     

Free energy profiles for H conduction in the D-pathway of Cytochrome c Oxidase: A study of the wild type and N98D mutant enzymes

The molecular mechanism for proton conduction in the D-pathway of Cytochrome c Oxidase (CcO) is investigated through the free energy profile, i.e., potential of mean force (PMF) calculations of both the native enzyme and the N98D mutant. The multistate empirical valence bond (MS-EVB) model was applied to simulate the interaction of an excess proton with the channel environment. In the study of the wild type enzyme, the PMF reveals the previously proposed proton trap inside the channel; it also shows a high free energy barrier against the passage of proton at the entry of the channel, where two conserved asparagines (ASN80/98) may be essential for the gating of proton uptake. We also present data from an investigation of the N98D mutant, which has been previously shown to completely eliminate proton pumping but significantly enhance the oxidase activity in Rhodobacter sphaeroides. These results suggest that mutating Asn98 to negatively charged aspartate will create an unfavorable energy barrier sufficiently high to prevent the overall proton uptake through the D-pathway, whereas with a protonated aspartic acid the proton conduction was found to be accelerated. Plausible explanations for the origin of the uncoupling of proton pumping from the oxidase activity will be discussed.     

Free energy profiles for H+ conduction in the D-pathway of Cytochrome c Oxidase: a study of the wild type and N98D mutant enzymes

The molecular mechanism for proton conduction in the D-pathway of Cytochrome c Oxidase (CcO) is investigated through the free energy profile, i.e., potential of mean force (PMF) calculations of both the native enzyme and the N98D mutant. The multistate empirical valence bond (MS-EVB) model was applied to simulate the interaction of an excess proton with the channel environment. In the study of the wild type enzyme, the PMF reveals the previously proposed proton trap inside the channel; it also shows a high free energy barrier against the passage of proton at the entry of the channel, where two conserved asparagines (ASN80/98) may be essential for the gating of proton uptake. We also present data from an investigation of the N98D mutant, which has been previously shown to completely eliminate proton pumping but significantly enhance the oxidase activity in Rhodobacter sphaeroides. These results suggest that mutating Asn98 to negatively charged aspartate will create an unfavorable energy barrier sufficiently high to prevent the overall proton uptake through the D-pathway, whereas with a protonated aspartic acid the proton conduction was found to be accelerated. Plausible explanations for the origin of the uncoupling of proton pumping from the oxidase activity will be discussed.     

Conformation and dynamics changes of bacteriorhodopsin and its D85N mutant in the absence of 2D crystalline lattice as revealed by site-directed C NMR

¹³C NMR spectra of [3-¹³C]Ala- and [1-¹³C]Val-labeled D85N mutant of bacteriorhodopsin (bR) reconstituted in egg PC or DMPC bilayers were recorded to gain insight into their secondary structures and dynamics. They were substantially suppressed as compared with those of 2D crystals, especially at the loops and several transmembrane α_II-helices. Surprisingly, the ¹³C NMR spectra of [3-¹³C]Ala-D85N turned out to be very similar to those of [3-¹³C]Ala-bR in lipid bilayers, in spite of the presence of globular conformational and dynamics changes in the former as found from 2D crystalline preparations. No further spectral change was also noted between the ground (pH 7) and M-like state (pH 10) as far as D85N in lipid bilayers was examined, in spite of their distinct changes in the 2D crystalline state. This is mainly caused by that the resulting ¹³C NMR peaks which are sensitive to conformation and dynamics changes in the loops and several transmembrane α_II-helices of the M-like state are suppressed already by fluctuation motions in the order of 10⁴-10⁵ Hz interfered with frequencies of magic angle spinning or proton decoupling. However, ¹³C NMR signal from the cytoplasmic α-helix protruding from the membrane surface is not strongly influenced by 2D crystal or monomer. Deceptively simplified carbonyl ¹³C NMR signals of the loop and transmembrane α-helices followed by Pro residues in [1-¹³C]Val-labeled bR and D85N in 2D crystal are split into two peaks for reconstituted preparations in the absence of 2D crystalline lattice. Fortunately, ¹³C NMR spectral feature of reconstituted [1-¹³C]Val and [3-¹³C]Ala-labeled bR and D85N was recovered to yield characteristic feature of 2D crystalline form in gel-forming lipids achieved at lowered temperatures.     

A longitudinal study of digit ratio (2D:4D) and other finger ratios in Jamaican children

It has been hypothesised that the ratio between the length of the 2nd and 4th digits (2D:4D) is a correlate of prenatal sex steroids, and this relationship is strongest for the right hand. Furthermore, it has been suggested that 2D:4D is sexually dimorphic, the dimorphism is determined early, and 2D:4D among children is stable with growth. Here, we present the first longitudinal study of right and left hand 2D:4D. Our sample was 108 (54 males) Jamaican children. The first measurements were made in 1998 when mean age was 9.68 +/- 1.39 years, and a second set of measurements were made in 2002. We found that: (i) there was a small increase in 2D:4D with age which was lowest in the right hand; (ii) 2D:4D was sexually dimorphic, the means for males and females differed in the same direction in the 1998 and 2002 samples, and the sex difference was significant in the 1998 but not in the 2002 sample; (iii) the correlation between the 1998 and 2002 measurements of 2D:4D was high, indicating that rank order of the ratio was stable across year groups; and (iv) the rate of change in 2D:4D did not differ significantly across year groups. We conclude that 2D:4D increases slightly with age in children with the effect less marked for the right hand (i.e. the hand which is likely to show the strongest association with prenatal steroids), 2D:4D is sexually dimorphic from an early age, and the rank order of 2D:4D is stable in children. We discuss the implications of our findings for the status of 2D:4D as a correlate of prenatal sex steroids. The patterns of change in other finger ratios are also considered.     

Genomic perspective on the photobiology of Halobacterium species NRC-1, a phototrophic,phototactic, and UV-tolerant haloarchaeon

Halobacterium species display a variety of responses to light, including phototrophic growth, phototactic behavior, and photoprotective mechanisms. The complete genome sequence of Halobacterium species NRC-1 (Proc Natl Acad Sci USA 97: 12176–12181, 2000), coupled with the availability of a battery of methods for its analysis makes this an ideal model system for studying photobiology among the archaea. Here, we review: (1) the structure of the 2.57 Mbp Halobacterium NRC-1 genome, including a large chromosome, two minichromosomes, and 91 transposable IS elements; (2) the purple membrane regulon, which programs the accumulation of large quantities of the light-driven proton pump, bacteriorhodopsin, and allows for a period of phototrophic growth; (3) components of the sophisticated pathways for color-sensitive phototaxis; (4) the gas vesicle gene cluster, which codes for cell buoyancy organelles; (5) pathways for the production of carotenoid pigments and retinal, (6) processes for the repair of DNA damage; and (7) putative homologs of circadian rhythm regulators. We conclude with a discussion of the power of systems biology for comprehensive understanding of Halobacterium NRC-1 photobiology. This revised version was published online in June 2006 with corrections to the Cover Date.     

Characterization of the N-terminal half-saturated state of calbindin D9k: NMR studies of the N56A mutant.

Calbindin D9k is a small EF-hand protein that binds two calcium ions with positive cooperativity. The molecular basis of cooperativity for the binding pathway where the first ion binds in the N-terminal site (1) is investigated by NMR experiments on the half-saturated state of the N56A mutant, which exhibits sequential yet cooperative binding (Linse S, Chazin WJ, 1995, Protein Sci 4:1038-1044). Analysis of calcium-induced changes in chemical shifts, amide proton exchange rates, and NOEs indicates that ion binding to the N-terminal binding loop causes significant changes in conformation and/or dynamics throughout the protein. In particular, all three parameters indicate that the hydrophobic core undergoes a change in packing to a conformation very similar to the calcium-loaded state. These results are similar to those observed for the (Cd2+)1 state of the wild-type protein, a model for the complementary half-saturated state with an ion bound in the C-terminal site (II). Thus, with respect to cooperativity in either of the binding pathways, binding of the first ion drives the conformation and dynamics of the protein far toward the (Ca2+)2 state, thereby facilitating binding of the second ion. Comparison with the half-saturated state of the analogous E65Q mutant confirms that mutation of this critical bidentate calcium ligand at position 12 of the consensus EF-hand binding loop causes very significant structural perturbations. This result has important implications regarding numerous studies that have utilized mutation of this critical residue for site deactivation.     

The study of CTLA-4 and vitamin D receptor polymorphisms in the Romanian type 1 diabetes population

Several studies suggested that part of the genetic susceptibility for Type 1 diabetes (TIDM) is encoded by some polymorphisms of CTLA-4 gene (2q33) and of Vitamin D Receptor gene (VDR; 12q12-14). Our aim was to assess their contribution to TIDM genetic susceptibility in the Romanian population. We typed CTLA-4 49 A/G and VDR Fok I (F/f), Apa I (A/a) and Taq I (T/t) polymorphisms by Sequence Specific Primer PCR (SSP-PCR) in 204 Romanian diabetic families (756 individuals: 212 TIDM probands and 544 unaffected parents and siblings). We studied alleles transmission using the Transmission Disequilibrium Test (TDT). We found an increased transmission of CTLA-4 49G allele to diabetics (54.8%, p=0.11). The transmission of F (56.1%, p=0.063), a (55.7%, p=0.061) and T (51.8%, p=0.37) alleles of VDR gene to diabetics was increased but did not reach statistical significance. In conclusion we found the same increased transmission of CTLA-4 49 G allele to diabetics as previously reported. VDR Foq I F allele seems to be predisposing while Taq I T allele seems to be protective.     

Clinical implementations of 4D pencil beam scanned particle therapy: Report on the 4D treatment planning workshop 2016 and 2017

In 2016 and 2017, the 8th and 9th 4D treatment planning workshop took place in Groningen (the Netherlands) and Vienna (Austria), respectively. This annual workshop brings together international experts to discuss research, advances in clinical implementation as well as problems and challenges in 4D treatment planning, mainly in spot scanned proton therapy. In the last two years several aspects like treatment planning, beam delivery, Monte Carlo simulations, motion modeling and monitoring, QA phantoms as well as 4D imaging were thoroughly discussed.This report provides an overview of discussed topics, recent findings and literature review from the last two years. Its main focus is to highlight translation of 4D research into clinical practice and to discuss remaining challenges and pitfalls that still need to be addressed and to be overcome.     

Exploring the effect of N308D mutation on protein tyrosine phosphatase-2 cause gain-of-function activity by a molecular dynamics study

One of the most common protein tyrosine phosphatase-2 (SHP2) mutations in Noonan syndrome is the N308D mutation, and it increases the activity of the protein. However, the molecular basis of the activation of N308D mutation on SHP2 conformations is poorly understood. Here, molecular dynamic simulations were performed on SHP2 and SHP2-N308D to explore the effect of N308D mutation on SHP2 cause gain of function activity, respectively. The principal component analysis, dynamic cross-correlation map, secondary structure analysis, residue interaction networks, and solvent accessible surface area analysis suggested that the N308D mutation distorted the residues interactions network between the allosteric site (residue Gly244-Gly246) and C-SH2 domain, including the hydrogen bond formation and the binding energy. Meanwhile, the activity of catalytic site (residue Gly503-Val505) located in the Q-loop in mutant increased due to this region's high fluctuations. Therefore, the substrate had more chances to access to the catalytic activity site of the precision time protocol domain of SHP2-N308D, which was easy to be exposed. In addition, we had speculated that the Lys244 located in the allosteric site was the key residue which lead to the protein conformation changes. Consequently, overall calculations presented in this study ultimately provide a useful understanding of the increased activity of SHP2 caused by the N308D mutation.     

2D:4D ratios in the first 2 years of life: Stability and relation to testosterone exposure and sensitivity

The relative lengths of the 2nd and 4th digits (2D:4D) may provide an easily measurable and stable anthropometric index of prenatal androgen exposure, but no study has examined the development of 2D:4D in infancy and the potential impact of neonatal testosterone levels. We collected 2D:4D ratios from 364 children between 0 and 2 years of age. Saliva samples were collected from 236 of these children 3 months after birth and analyzed for testosterone. In addition, 259 children provided DNA samples which were genotyped for the CAG repeat polymorphism in the androgen receptor. There was substantial variability across age in 2D:4D. Sex differences were small compared to adults and did not consistently reach statistical significance. This suggests that 2D:4D may not function well as a proxy measure of prenatal testosterone exposure in infancy. In addition, the interaction of salivary T and CAG repeats predicted right hand digit ratio at 12 months and left hand digit ratio at 12 months and 24 months in males. The interaction of salivary testosterone and CAG repeat length also predicted change in left hand 2D:4D from 2 weeks to 12 months in males. This suggests that 2D:4D in adults may reflect, in part, neonatal testosterone exposure. No significant relationships were observed within females. No significant relationships were observed when salivary testosterone and CAG repeats were examined independent of each other. Results have important implications for the design and interpretation of studies which use 2D:4D as a proxy measure of prenatal testosterone exposure.     

Cyclic nucleotide phosphodiesterases in larval brain of wild type and dunce mutant strains of Drosophila melanogaster: isoenzyme pattern and activation by Ca2+/calmodulin

Like adult heads and whole flies, larval brains of wild type Drosophila melanogaster contain two major soluble cyclic nucleotide phosphodiesterases, forms I and II. Larval brains of the learning-defective mutant strain, dunceM11, contain only the form I enzyme. In both wild type and dunce strains the form I enzyme is activated by Ca2+/calmodulin. A time-dependent loss of this Ca2+ activation was observed.     

Challenges of radiotherapy: Report on the 4D treatment planning workshop 2013

This report, compiled by experts on the treatment of mobile targets with advanced radiotherapy, summarizes the main conclusions and innovations achieved during the 4D treatment planning workshop 2013. This annual workshop focuses on research aiming to advance 4D radiotherapy treatments, including all critical aspects of time resolved delivery, such as in-room imaging, motion detection, motion managing, beam application, and quality assurance techniques. The report aims to revise achievements in the field and to discuss remaining challenges and potential solutions. As main achievements advances in the development of a standardized 4D phantom and in the area of 4D-treatment plan optimization were identified. Furthermore, it was noticed that MR imaging gains importance and high interest for sequential 4DCT/MR data sets was expressed, which represents a general trend of the field towards data covering a longer time period of motion. A new point of attention was work related to dose reconstructions, which may play a major role in verification of 4D treatment deliveries. The experimental validation of results achieved by 4D treatment planning and the systematic evaluation of different deformable image registration methods especially for inter-modality fusions were identified as major remaining challenges. A challenge that was also suggested as focus for future 4D workshops was the adaptation of image guidance approaches from conventional radiotherapy into particle therapy. Besides summarizing the last workshop, the authors also want to point out new evolving demands and give an outlook on the focus of the next workshop.     

Lipopolysaccharide ofRhodospirillum salinarum 40: structural studies on the core and lipid A region

The structural elucidation of lipid A of the cell wall lipopolysaccharide (LPS) ofRhodospirillum salinarum 40 by chemical methods and laser desorption mass spectrometry revealed the presence of a mixed lipid A composed of three different 1,4 bisphosphorylated β(1→6)-linked backbone hexosaminyl-hexosamine disaccharides, i.e. those composed of GlCN→GlcN, 2,3-diamino-2,3-dideoxy-d-Glc-(DAG)→DAG, and DAG→GlcN. Lipid A ofR. salinarum contained preferentially 3-OH-18:0 and 3-OH-14:0 as amide-linked andcisΔ¹¹-18:1 and c19:0 as ester-linked fatty acids. The mass spectra of the liberated acyl-oxyacyl residues proved the concomitant presence of 3-O-(cisΔ¹¹-18:1)-18:0 and 3-O-(c19:0)-14:0 as the predominating diesters in this mixed lipid A. The glycosidically linked and the ester-linked phosphate groups of the backbone disaccharide were neither substituted by ethanolamine phosphorylethanolamine, nor by 4-amino-4-deoxy-l-arabinose, in contrast to most of the enterobacterial lipid As. In the core oligosaccharide fraction, a HexA (1→4)HexA(1→5)Kdo-trisaccharide was identified by methylation analysis. The terminal HexA (hexuronic acid) is possibly 4-OMe-GalA, a component described here as an LPS constituent for the first time. LPS ofR. salinarum showed a lethality in C57BL/10 ScSN (LPS-responder)-mice) of an order of 10⁻¹–10⁻² of that reported forSalmonella abortus equi LPS, and it was also capable of inducing TNFα and IL6 in macrophages of C57BL/10ScSN mice.     

C、N、P对杜氏盐藻突变藻株Zea1生长和积累玉米黄素的影响

通过UV辐照和NTG诱变处理杜氏盐藻野生型藻株得到一株杜氏盐藻高产玉米黄素突变株Zea1,以此突变藻株为实验材料,系统研究了光照强度、盐浓度、碳源、氮源、磷源等对Zea1生长和玉米黄素积累合成的影响。葡萄糖在10mmol/L时既适合Zea1生长,又有利于其玉米黄素的积累。KNO3浓度为1mmol/L时最适合突变株藻细胞生长,而(NH4)2SO4浓度为1mmol/L最有利于藻细胞内玉米黄素的积累。综合来看,1mmol/L(NH4)2SO4为最优氮源。KH2PO4浓度为0.1mmol/L时Zea1藻细胞积累玉米黄素含量最高,同时在此浓度下也最适宜藻细胞的生长。讨论了此结果的机理和意义,为利用杜氏盐藻大规模生产玉米黄素提供了理论依据。     

Open questions on the 3D structures of collagen containing vertebrate mineralized tissues: A perspective

Our current understanding of the structures of vertebrate mineralized tissues is largely based on light microscopy/histology and projections of 3D structures onto 2D planes using electron microscopy. We know little about the fine details of these structures in 3D at the length scales of their basic building blocks, the inherent variations of structure within a tissue and the cell-extracellular tissue interfaces. This limits progress in understanding tissue formation, relating structure to mechanical and metabolic functions, and obtaining deeper insights into pathologies and the evolution of these tissues. In this perspective we identify and discuss a series of open questions pertaining to collagen containing vertebrate mineralized tissues that can be addressed using appropriate 3D structural determination methods. By so doing we hope to encourage more research into the 3D structures of mineralized vertebrate tissues.