Carbohydrate moiety of the Petunia inflata S3 protein is not required for self-incompatibility interactions between pollen and pistil.

For Petunia inflata and Nicotiana alata, which display gametophytic self-incompatibility, S proteins (the products of the multiallelic S gene in the pistil) have been shown to control the pistil's ability to recognize and reject self-pollen. The biochemical mechanism for rejection of self-pollen by S proteins has been shown to involve their ribonuclease activity; however, the molecular basis for self/non-self recognition by S proteins is not yet understood. Here, we addressed whether the glycan chain of the S3 protein of P. inflata is involved in self/non-self recognition by producing a nonglycosylated S3 protein in transgenic plants and examining the effect of deglycosylation on the ability of the S3 protein to reject S3 pollen. The S3 gene was mutagenized by replacing the codon for Asn-29, which is the only potential N-glycosylation site of the S3 protein, with a codon for Asp, and the mutant S3 gene was introduced into P. inflata plants of the S1S2 genotype. Six transgenic plants that produced a normal level of the nonglycosylated S3 protein acquired the ability to reject S3 pollen completely. These results suggest that the carbohydrate moiety of the S3 protein does not play a role in recognition or rejection of self-pollen and that the S allele specificity determinant of the S3 protein and those S proteins that contain a single glycan chain at the same site as the S3 protein must reside in the amino acid sequence itself.     

Localization of a beta-crystallin gene, Hu beta A3/A1 (gene symbol: CRYB1), to the long arm of human chromosome 17

We have assigned a human beta-crystallin gene, Hu beta A3/A1 (gene symbol: CRYB1), to chromosome 17 using a panel of 19 human-hamster somatic cell hybrids and blot-hybridization analysis of cell hybrid DNA. Positive probe-hybridization signal was detected in a hybrid that had lost the short arm of human chromosome 17 but retained the long arm, translocated to a hamster chromosome. In addition, in situ hybridization analysis of metaphase chromosome spreads of this cell line suggested that the most probable location for CRYB1 is on the long arm of chromosome 17, in the region q21.     

APP processing is regulated by cytoplasmic phosphorylation

Amyloid-beta peptide (Abeta) aggregate in senile plaque is a key characteristic of Alzheimer's disease (AD). Here, we show that phosphorylation of amyloid precursor protein (APP) on threonine 668 (P-APP) may play a role in APP metabolism. In AD brains, P-APP accumulates in large vesicular structures in afflicted hippocampal pyramidal neurons that costain with antibodies against endosome markers and the beta-secretase, BACE1. Western blot analysis reveals increased levels of T668-phosphorylated APP COOH-terminal fragments in hippocampal lysates from many AD but not control subjects. Importantly, P-APP cofractionates with endosome markers and BACE1 in an iodixanol gradient and displays extensive colocalization with BACE1 in rat primary cortical neurons. Furthermore, APP COOH-terminal fragments generated by BACE1 are preferentially phosphorylated on T668 verses those produced by alpha-secretase. The production of Abeta is significantly reduced when phosphorylation of T668 is either abolished by mutation or inhibited by T668 kinase inhibitors. Together, these results suggest that T668 phosphorylation may facilitate the BACE1 cleavage of APP to increase Abeta generation.     

Effects of a HP0859 (rfaD) knockout mutation on lipopolysaccharide structure of Helicobacter pylori 26695 and the bacterial adhesion on AGS cells

Lipopolysaccharide (LPS) is considered as an important virulence factor of Helicobacter pylori, and contributes to infection persistence and disease severity. ADP-l-glycero-d-manno-heptose-6-epimerase is an enzyme essential for LPS synthesis and understanding of its biochemistry is critical for drug development. We cloned one putative ortholog of Escherichia colirfaD, HP0859, from H. pylori 26695. Determination of the native molecular weight of the recombinant HP0859 protein suggests that the protein is likely a hexamer. NADP⁺, instead of NAD⁺, was proved to be the physiological cofactor for HP0859 protein. Circular dichroism spectrum analysis demonstrated that the secondary structure of this protein is significantly altered when the cofactor is removed. We also constructed an HP0859 knockout mutant and examined its phenotypic properties. The HP0859 knockout mutant exhibited a severe truncation of LPS, a decreased growth rate, and a higher susceptibility to novobiocin. Disruption of HP0859 also reduced the adhesive capacity of H. pylori to AGS cells, and the infected cells failed to display the classic hummingbird phenotype. Complementation of the HP0859 knockout mutation restored these phenotypes completely. In conclusion, we demonstrate that HP0859 codes for a protein essential for the LPS inner core biosynthesis in H. pylori and an intact LPS structure contributes to the adherence ability of this bacterium.     

Chemical reactivity of brome mosaic virus capsid protein

Viral particles are biological machines that have evolved to package, protect, and deliver the viral genome into the host via regulated conformational changes of virions. We have developed a procedure to modify lysine residues with S-methylthioacetimidate across the pH range from 5.5 to 8.5. Lysine residues that are not completely modified are involved in tertiary or quaternary structural interactions, and their extent of modification can be quantified as a function of pH. This procedure was applied to the pH-dependent structural transitions of brome mosaic virus (BMV). As the reaction pH increases from 5.5 to 8.5, the average number of modified lysine residues in the BMV capsid protein increases from 6 to 12, correlating well with the known pH-dependent swelling behavior of BMV virions. The extent of reaction of each of the capsid protein's lysine residues has been quantified at eight pH values using coupled liquid chromatography-tandem mass spectrometry. Each lysine can be assigned to one of three structural classes identified by inspection of the BMV virion crystal structure. Several lysine residues display reactivity that indicates their involvement in dynamic interactions that are not obvious in the crystal structure. The influence of several capsid protein mutants on the pH-dependent structural transition of BMV has also been investigated. Mutant H75Q exhibits an altered swelling transition accompanying solution pH increases. The H75Q capsids show increased reactivity at lysine residues 64 and 130, residues distal from the dimer interface occupied by H75, across the entire pH range.     

Time-resolved fluorometric and differential scanning calorimetric investigation of dehydroergosterol in 1-stearoyl-2-caprylphosphatidylcholine bilayers

Thermal and dynamic properties of dehydroergosterol (DHE) in 1-stearoyl-2-capryl-sn-glycero-3-phosphocholine [C(18):C(10)PC] have been studied by differential scanning calorimetry (DSC) and multifrequency phase-modulation fluorometry. C(18):C(10)PC is an asymmetric mixed-chain phosphatidylcholine known to form highly ordered mixed interdigitated bilayers below the maximal transition temperature, Tm, and partially interdigitated bilayers above Tm. This lipid system is thus unique in assessing the interactions between sterols and interdigitated lipid bilayers. DHE is a fluorescent analogue of cholesterol shown in previous studies to behave like cholesterol in noninterdigitated symmetric diacylphosphatidylcholines. DSC data show that DHE exhibits similar characteristics to cholesterol [Chong & Choate (1989) Biophys. J. 55, 551-556] in C(18):C(10)PC bilayers. DHE abolishes the phase transition of C(18):C(10)PC at 27 mol % compared to 25 mol % for cholesterol and decreases Tm, the onset temperature (To), and the completion temperature (Tc), at a similar rate to cholesterol at about -0.25 degrees C per mole percent DHE. Fluorescence data show that the rotational motion of DHE can be described by a hindered anisotropic model. In the gel state of C(18):C(10)PC, the rotational correlation of DHE decreases monotonically with increasing DHE content up to 24 mol %, suggesting that DHE causes a disordering/spacing effect on the packing of mixed interdigitated C(18):C(10)PC bilayers. The rotational correlation time undergoes an abrupt increase from 24 to 27 mol % DHE. Abrupt changes in the DSC parameters were also observed in the neighborhood of 27 mol %, suggesting that major reorganization takes place around this concentration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dipsticking the major groove of DNA with enzymatically incorporated spin-labeled deoxyuridines by electron spin resonance spectroscopy

Site-specifically spin-labeled deoxyuridine triphosphates with tethers of different lengths were synthesized and then enzymatically incorporated with terminal transferase to form a spin-labeled poly(dT) copolymer. The spin-labeled copolymers were annealed with poly(dA) to form a duplex, which was analyzed by electron spin resonance spectroscopy in a solution of low ionic strength. The spin labels are attached in position 5 of the deoxyuridine and protrude into the major groove. Based on the correlation between tether length of the spin label and the electron spin resonance lineshape, we show that the depth of the major groove of a DNA in its B-form is about 8 A in solution, which is in good agreement with X-ray fiber studies. We also conclude, based on electron spin resonance lineshape simulation data, that the correlation time of the bases in a DNA duplex is of the order of nanoseconds.     

Heme oxygenase is involved in nitric oxide- and auxin-induced lateral root formation in rice

Lateral root (LR) development performs the essential tasks of providing water, nutrients, and physical support to plants. Therefore, understanding the regulation of LR development is of agronomic importance. In this study, we examined the effect of nitric oxide (NO), auxin, and hemin (Hm) on LR formation in rice. Treatment with Hm [a highly effective heme oxygenase (HO) inducer], sodium nitroprusside (SNP, an NO donor), or indole-3-butyric acid (IBA, a naturally occurring auxin) induced LR formation and HO activity. LR formation and HO activity induced by SNP and IBA but not Hm was reduced by the specific NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide. As well, Hm, SNP, and IBA could induce OsHO1 mRNA expression. Zn protoporphyrin IX (the specific inhibitor of HO) and hemoglobin (the carbon monoxide/NO scavenger) reduced LR number and HO activity induced by Hm, SNP, and IBA. Our data suggest that HO is required for Hm-, auxin-, and NO-induced LR formation in rice.