Investigating DNA Radiation Damage Using X-Ray Absorption Spectroscopy

The biological influence of radiation on living matter has been studied for years; however, several questions about the detailed mechanism of radiation damage formation remain largely unanswered. Among all biomolecules exposed to radiation, DNA plays an important role because any damage to its molecular structure can affect the whole cell and may lead to chromosomal rearrangements resulting in genomic instability or cell death. To identify and characterize damage induced in the DNA sugar-phosphate backbone, in this work we performed x-ray absorption spectroscopy at the P K-edge on DNA irradiated with either UVA light or protons. By combining the experimental results with theoretical calculations, we were able to establish the types and relative ratio of lesions produced by both UVA and protons around the phosphorus atoms in DNA.     

X-Ray Absorption Spectroscopy of Dinuclear Metallohydrolases

In this mini-review, we briefly discuss the physical origin of x-ray absorption spectroscopy (XAS) before illustrating its application using dinuclear metallohydrolases as exemplary systems. The systems we have selected for illustrative purposes present a challenging problem for XAS, one that is ideal to demonstrate the potential of this methodology for structure/function studies of metalloenzymes in general. When the metal ion is redox active, XAS provides a sensitive measure of oxidation-state-dependent differences. When the metal ion is zinc, XAS is the only spectroscopic method that will provide easily accessible structural information in solution. In the case of heterodimetallic sites, XAS has the unique ability to interrogate each metal site independently in the same sample. One of the strongest advantages of XAS is its ability to examine metal ion site structures with crystallographic precision, without the need for a crystal. This is key for studying flexible metal ion sites, such as those described in the selected examples, because it allows one to monitor structural changes that occur during substrate turnover.     

X-Ray Absorption Spectroscopy of Dinuclear Metallohydrolases

In this mini-review, we briefly discuss the physical origin of x-ray absorption spectroscopy (XAS) before illustrating its application using dinuclear metallohydrolases as exemplary systems. The systems we have selected for illustrative purposes present a challenging problem for XAS, one that is ideal to demonstrate the potential of this methodology for structure/function studies of metalloenzymes in general. When the metal ion is redox active, XAS provides a sensitive measure of oxidation-state-dependent differences. When the metal ion is zinc, XAS is the only spectroscopic method that will provide easily accessible structural information in solution. In the case of heterodimetallic sites, XAS has the unique ability to interrogate each metal site independently in the same sample. One of the strongest advantages of XAS is its ability to examine metal ion site structures with crystallographic precision, without the need for a crystal. This is key for studying flexible metal ion sites, such as those described in the selected examples, because it allows one to monitor structural changes that occur during substrate turnover.     

Determination of Cu Environments in the Cyanobacterium Anabaena flos-aquae by X-Ray Absorption Spectroscopy

Whole cells and peptidoglycan isolated from cell walls of the cyanobacterium Anabaena flos-aquae were lyophilized and used at pH 2 and pH 5 in Cu(II) binding studies. X-ray absorption spectra measured at the Cu K-edge were used to determine the oxidation states and chemical environments of Cu species in the whole-cell and peptidoglycan samples. In the whole-cell samples, most of the Cu retained at both pH values was coordinated by phosphate ligands. The whole-cell fractions contained significant concentrations of Cu(I) as well as Cu(II). An X-ray absorption near-edge spectrum analysis suggested that Cu(I) was coordinated by amine and thiol ligands. An analysis of the peptidoglycan fractions found that more Cu was adsorbed by the peptidoglycan fraction prepared at pH 5, due to increased chelation by amine and carboxyl ligands. The peptidoglycan fractions, also referred to as the cell wall fractions, contained little or no Cu(I). The Cu loading level was 30 times higher in the cell wall sample prepared at pH 5 than in the sample prepared at pH 2. Amine and bidentate carboxyl ligands had similar relative levels of importance in cell wall peptidoglycan samples prepared at both pH values, but phosphate coordination was insignificant.     

金属叶绿素a配位结构的研究

在丙酮溶液中合成得到镧-叶绿素a、钐-叶绿素a和铜-叶绿素a复合物,并研究了它们的紫外可见光谱（UV-Vis）、Fourier红外光谱（FT-IR）和EXAFS结构.镧-叶绿素a、钐-叶绿素a和铜-叶绿素a的UV-Vis谱、FT-IR谱与叶绿素a（含镁）的光谱性质相似,但与脱镁叶绿素a的光谱性质差异很大.证明了La³⁺、Sm³⁺、Cu²⁺已配位到脱镁叶绿素的卟啉环上,形成了镧-叶绿素a、钐-叶绿素a和铜-叶绿素a复合物.通过扩展X射线吸收精细结构谱(EXAFS)研究表明：合成镧-叶绿素a、钐-叶绿素a具有双层夹心结构.La(Ⅲ)、Sm(Ⅲ)夹于两个卟啉环之间, 与上下卟啉环上共八个N原子配位, La-N平均键长0.261 nm,Sm-N平均键长0.243 nm, 而铜-叶绿素a的EXAFS表明为一单层结构,Cu(Ⅱ)与卟啉环中的四个N原子配位,Cu-N平均键长0.197 nm.元素分析也证明镧-叶绿素a、钐-叶绿素a为双层结构,铜-叶绿素a为单层结构.     

X-Ray Absorption Near-Edge Structure (XANES) Spectroscopy Study of the Interaction of Silver Ions with Staphylococcus aureus,Listeria monocytogenes,and Escherichia coli

Silver ions are widely used as antibacterial agents, but the basic molecular mechanism of this effect is still poorly understood. X-ray absorption near-edge structure (XANES) spectroscopy at the Ag LIII, S K, and P K edges reveals the chemical forms of silver in Staphylococcus aureus and Escherichia coli (Ag⁺ treated). The Ag LIII-edge XANES spectra of the bacteria are all slightly different and very different from the spectra of silver ions (silver nitrate and silver acetate), which confirms that a reaction occurs. Death or inactivation of bacteria was observed by plate counting and light microscopy. Silver bonding to sulfhydryl groups (Ag-S) in cysteine and Ag-N or Ag-O bonding in histidine, alanine, and dl-aspartic acid was detected by using synthesized silver-amino acids. Significantly lower silver-cysteine content, coupled with higher silver-histidine content, in Gram-positive S. aureus and Listeria monocytogenes cells indicates that the peptidoglycan multilayer could be buffering the biocidal effect of silver on Gram-positive bacteria, at least in part. Bonding of silver to phosphate groups was not detected. Interaction with DNA or proteins can occur through Ag-N bonding. The formation of silver-cysteine can be confirmed for both bacterial cell types, which supports the hypothesis that enzyme-catalyzed reactions and the electron transport chain within the cell are disrupted.     

A Study of the DNA Structure in Films Using FTIR Spectroscopy

Biophysics - The aqueous environment and ionic surroundings are the most important factors that determine the conformation of DNA and its functioning in the cell. The specificity of the interaction...     

Electron Microscope Observations on the Fine Structure of Parietal Cells

An electron microscope study has been made of the structure of parietal cells in cats, dogs, and rats and of the cells lining the gastric glands of Bufo spinulosus. It is characteristic of all these cells to contain numerous vesicles about 0.05 to 0.3 µ in size. In the mammalian parietal cells an intracellular system of canaliculi is also observed, which is much more complex in the rat than in the cat or dog. Stimulation with histamine causes in the cat a very marked hypertrophy of the canalicular system with development of a large number of villi and a decrease in the number of vesicles. In Bufo, histamine induces the formation of a very complex system of membrane infoldings which circumscribe finger-like processes that entirely fill the glandular lumen. The cytoplasmic vesicles diminish or disappear. These experiments show that under histamine stimulation all these cells undergo a great increase in the cell membrane area. These findings provide additional circumstantial evidence that parietal cells play a role in the secretion of hydrochloric acid.     

Substrate Specificity of the Highly Alkalophilic Bacterial Proteinase Esperase: Relation to the X-Ray Structure

Esperase is a highly alkalophilic bacterial proteinase produced by Bacillus lentus. The enzyme hydrolyzes peptide bonds comprising the carboxylic groups of hydrophobic as well as hydrophilic residues in the oxidized insulin B chain. Some of these bonds are not attacked by other alkaline microbial proteinases. P1-P4 specificity was determined by a series of peptide nitroanilides. The S1 recognition loop exhibits a preference for Phe. The "cleft" of the smallest subsite S2 prefers Ala and exhibits low affinity for the larger chain of Leu. S3 is more open than the other subsites and can accept a variety of residues. Hydrophobic interactions predominate in the S4-P4 interactions because S4 can accommodate Phe very well. The results characterize Esperase as an endopeptidase with a broader specificity in comparison with other microbial serine proteinases. This is probably owing to a more flexible substrate binding site.     

The Fine Structure of Leaf Cells of Copper-deficient Oats

Electron micrographs of copper-deficient oat leaves showed onlyminor structural differences from normal oat leaves in thatthere were reduced numbers of compartments in the chloroplaststacks with no differences in stromacentres, plastoglobuli,mitochondria, nuclei, or cell walls. The cells of the whitewithered tip characteristic of copper-deficient oat leaves werefound to be full of fibrous masses.     

Measuring the Bending Stiffness of Bacterial Cells Using an Optical Trap

We developed a protocol to measure the bending rigidity of filamentous rod-shaped bacteria. Forces are applied with an optical trap, a microscopic three-dimensional spring made of light that is formed when a high-intensity laser beam is focused to a very small spot by a microscope''s objective lens. To bend a cell, we first bind live bacteria to a chemically-treated coverslip. As these cells grow, the middle of the cells remains bound to the coverslip but the growing ends are free of this restraint. By inducing filamentous growth with the drug cephalexin, we are able to identify cells in which one end of the cell was stuck to the surface while the other end remained unattached and susceptible to bending forces. A bending force is then applied with an optical trap by binding a polylysine-coated bead to the tip of a growing cell. Both the force and the displacement of the bead are recorded and the bending stiffness of the cell is the slope of this relationship.Download video file.^{(65M, mp4)}     

Changes in the Fine Structure of Microbial Cells Induced by Chaotropic Salts

The electron microscopic examination of thin sections of cells of the yeasts Saccharomyces cerevisiae and Pichia pastoris and the gram-positive bacteria Micrococcus luteus and Bacillus subtilis showed that cell treatment with the chaotropic salts guanidine hydrochloride (6 M) and guanidine thiocyanate (4 M) at 37°C for 3–5 h or at 100°C for 5–6 min induced degradative processes, which affected almost all cellular structures. The cell wall, however, retained its ultrastructure, integrity, and rigidity, due to which the morphology of cells treated with the chaotropic salts did not change. High-molecular-weight DNA was localized in a new cell compartment, the ectoplasm (a peripheral hydrophilic zone). The chaotropic salts destroyed the outer and inner membranes and partially degraded the outer and inner protein coats of Bacillus subtilis spores, leaving their cortex (the murein layer) unchanged. The spore core became accessible to stains and showed the presence of regions with high and low electron densities. The conditions of cell treatment with the chaotropic salts were chosen to provide for efficient in situ PCR analysis of the 16S and 18S rRNA genes with the use of oligonucleotide primers.     

X射线衍射法在研究蛋白质动态过程中的应用

介绍了X射线衍射技术在研究蛋白质动态过程中的应用.首先介绍了用常规X射线衍射法和劳埃X射线衍射法等数据采样法研究反应时间为几分钟的蛋白质催化反应.然后介绍了通过选择不匹配底物,不适宜酸度,选择温度和酸度的跳跃,金属和光化学瞬时激发达到反应的同步来研究反应时间为几秒钟的蛋白质催化反应.     

Fine Structure of Penetration of Cultured Cells by Isospora canis Sporozoites

SYNOPSIS Monolayers of Embryonic Bovine Trachea (EBTr) cells were inoculated with Isospora canis Nemeséri spcrozoites. As penetration commenced, they were fixed, stained with OsO₄-ruthenium red, dehydrated, embedded and sectioned in situ. Examination by electron microscopy revealed that host cell membranes remained intact during penetration. The sporozoites caused an invagination of the cell's plasmalemma until the parasites were entirely within the cell, after which the invagination was sealed by short pseudopodia enclosing the parasite within a membrane-lined vacuole inside the cells. Rhoptries and micronemes, which appeared as branched elements of the same network, became less tortuous near the conoid and often became empty or partially empty during penetration. Concurrent with the appearance of these partially empty rhoptries, vesiculations were seen in the host cell cytoplasm opposite the apical tip of the sporczoite. Constrictions of the sporozoite during entry were probably due to bands of microfilaments beneath the plasmalemma and elsewhere in the cytoplasm of the host cell.     

Differential Damage in Bacterial Cells by Microwave Radiation on the Basis of Cell Wall Structure

Microwave radiation in Escherichia coli and Bacillus subtilis cell suspensions resulted in a dramatic reduction of the viable counts as well as increases in the amounts of DNA and protein released from the cells according to the increase of the final temperature of the cell suspensions. However, no significant reduction of cell density was observed in either cell suspension. It is believed that this is due to the fact that most of the bacterial cells inactivated by microwave radiation remained unlysed. Scanning electron microscopy of the microwave-heated cells revealed severe damage on the surface of most E. coli cells, yet there was no significant change observed in the B. subtilis cells. Microwave-injured E. coli cells were easily lysed in the presence of sodium dodecyl sulfate (SDS), yet B. subtilis cells were resistant to SDS.     

Fine Structure Recombinational Analysis of Cloned Genes Using Yeast Transformation

We describe a general method for analyzing the genetic fine structure of plasmid-borne genes in yeast. Previously we had reported that a linearized plasmid is efficiently rescued by recombination with a homologous restriction fragment when these are co-introduced by DNA-mediated transformation of yeast. Here, we show that a mutation can be localized to a small DNA interval when members of a deletion series of wild-type restriction fragments are used in the rescue of a linearized mutant plasmid. The resolution of this method is to at least 30 base pairs and is limited by the loss of a wild-type marker with proximity to a free DNA end. As a means for establishing the nonidentity of two mutations, we determined the resolution of two-point crosses with a mutant linearized plasmid and a mutant homologous restriction fragment. Recombination between mutations separated by as little as 100 base pairs was detected. Moreover, the results indicate that exchange within a marked interval results primarily from one of two single crossovers that repair the linearized plasmid. These approaches to mapping the genetic fine structure of plasmids should join existing methods in a robust approach to the mutational analysis of gene structure in yeast.