Crystallographic structure reveals phosphorylated pilin from Neisseria: phosphoserine sites modify type IV pilus surface chemistry and fibre morphology

Understanding the structural biology of type IV pili, fibres responsible for the virulent attachment and motility of numerous bacterial pathogens, requires a detailed understanding of the three-dimensional structure and chemistry of the constituent pilin subunit. X-ray crystallographic refinement of Neisseria gonorrhoeae pilin against diffraction data to 2.6 A resolution, coupled with mass spectrometry of peptide fragments, reveals phosphoserine at residue 68. Phosphoserine is exposed on the surface of the modelled type IV pilus at the interface of neighbouring pilin molecules. The site-specific mutation of serine 68 to alanine showed that the loss of the phosphorylation alters the morphology of fibres examined by electron microscopy without a notable effect on adhesion, transformation, piliation or twitching motility. The structural and chemical characterization of protein phosphoserine in type IV pilin subunits is an important indication that this modification, key to numerous regulatory aspects of eukaryotic cell biology, exists in the virulence factor proteins of bacterial pathogens. These O-linked phosphate modifications, unusual in prokaryotes, thus merit study for possible roles in pilus biogenesis and modulation of pilin chemistry for optimal in vivo function.     

Prevention and correction of temporal hair loss in rhytidectomy

Routine incisions in the temporal area for rhytidectomy often remove hair-bearing skin anterior to the ear. This results in a cosmetic deformity, making the surgical intervention clearly visible. This is especially problematic for revision rhytidectomy or for patients with naturally high hairlines. This article describes a systematic approach to the temporal hairline and introduces a novel, hair-bearing, transposition flap that corrects iatrogenic loss of the preauricular tuft of hair.     

Molecular mechanism of voltage sensor movements in a potassium channel

Voltage-gated potassium channels are six-transmembrane (S1-S6) proteins that form a central pore domain (4 x S5-S6) surrounded by four voltage sensor domains (S1-S4), which detect changes in membrane voltage and control pore opening. Upon depolarization, the S4 segments move outward carrying charged residues across the membrane field, thereby leading to the opening of the pore. The mechanism of S4 motion is controversial. We have investigated how S4 moves relative to the pore domain in the prototypical Shaker potassium channel. We introduced pairs of cysteines, one in S4 and the other in S5, and examined proximity changes between each pair of cysteines during activation, using Cd2+ and copper-phenanthroline, which crosslink the cysteines with metal and disulphide bridges, respectively. Modelling of the results suggests a novel mechanism: in the resting state, the top of the S3b-S4 voltage sensor paddle lies close to the top of S5 of the adjacent subunit, but moves towards the top of S5 of its own subunit during depolarization--this motion is accompanied by a reorientation of S4 charges to the extracellular phase.     

Accurate detection of aneuploidies in array CGH and gene expression microarray data

MOTIVATION: Chromosomal copy number changes (aneuploidies) are common in cell populations that undergo multiple cell divisions including yeast strains, cell lines and tumor cells. Identification of aneuploidies is critical in evolutionary studies, where changes in copy number serve an adaptive purpose, as well as in cancer studies, where amplifications and deletions of chromosomal regions have been identified as a major pathogenetic mechanism. Aneuploidies can be studied on whole-genome level using array CGH (a microarray-based method that measures the DNA content), but their presence also affects gene expression. In gene expression microarray analysis, identification of copy number changes is especially important in preventing aberrant biological conclusions based on spurious gene expression correlation or masked phenotypes that arise due to aneuploidies. Previously suggested approaches for aneuploidy detection from microarray data mostly focus on array CGH, address only whole-chromosome or whole-arm copy number changes, and rely on thresholds or other heuristics, making them unsuitable for fully automated general application to gene expression datasets. There is a need for a general and robust method for identification of aneuploidies of any size from both array CGH and gene expression microarray data. RESULTS: We present ChARM (Chromosomal Aberration Region Miner), a robust and accurate expectation-maximization based method for identification of segmental aneuploidies (partial chromosome changes) from gene expression and array CGH microarray data. Systematic evaluation of the algorithm on synthetic and biological data shows that the method is robust to noise, aneuploidal segment size and P-value cutoff. Using our approach, we identify known chromosomal changes and predict novel potential segmental aneuploidies in commonly used yeast deletion strains and in breast cancer. ChARM can be routinely used to identify aneuploidies in array CGH datasets and to screen gene expression data for aneuploidies or array biases. Our methodology is sensitive enough to detect statistically significant and biologically relevant aneuploidies even when expression or DNA content changes are subtle as in mixed populations of cells. AVAILABILITY: Code available by request from the authors and on Web supplement at http://function.cs.princeton.edu/ChARM/     

Influence of Environmental Salinity on Messenger RNA Levels of Growth Hormone, Prolactin, and Somatolactin in Pituitary of the Channel Catfish (Ictalurus punctatus)

Pituitary growth hormone (GH), prolactin (PRL), and somatolactin (SL) messenger RNA levels in channel catfish (Ictalurus punctatus) were examined under various environmental and physiological conditions. Catfish were sampled following salinity challenge, during the winter (December) and spring or summer (April or July), and at different sizes (15–18 g, 620–664 g, and 956–1134 g). When catfish (956–1134 g) were transferred from freshwater to saline water containing 8 ppt NaCl, their plasma [Na⁺] increased significantly above values in the freshwater control group until they were transferred back to freshwater. Pituitary GH mRNA levels were low for the first 24 hours following transfer to saline water, but thereafter were significantly elevated above control values until the fish were transferred back to freshwater. Pituitary GH mRNA levels were highest in July and lowest in December. Growth hormone mRNA levels were also elevated in the size groups 15–18 g and 956–1134 g in July when compared with December values. Pituitary PRL mRNA levels increased for the first 24 hours following transfer to saline water (956–1134 g), but thereafter were significantly lower than control values until the fish were transferred back to freshwater. Pituitary PRL mRNA levels were highest in April and July and lowest in December, and were also elevated in the size groups 620–664 g and 956–1134 g. Pituitary SL mRNA levels were unaffected in catfish transferred to saline water; however, levels were significantly elevated in catfish of the 956–1134-g size group sampled in April when compared with December. These results suggest the involvement of GH in adaptation to brackish water and of PRL in adaptation to freshwater in the catfish, and seasonal and size-related differences in pituitary GH, PRL, and SL mRNA levels. Received May 17, 2000; accepted October 30, 2000     

Effect of Rainbow Trout Growth Hormone Complementary DNA on Body Shape,Carcass Yield,and Carcass Composition of F1 and F2 Transgenic Common Carp (Cyprinus carpio

The effect of rainbow trout growth hormone complementary DNA on body shape, dress-out yield, and body composition were assessed in the F1 and F2 generations of transgenic common carp (Cyprinus carpio). All measurements were compared with those for nontransgenic full-sibling common carp in their respective families, and the fish were communally evaluated in earthen ponds. The body weight and length were highly correlated (P <0.01) in both genotypes in all the families. Head morphometrics were negatively correlated (P <0.05) to weight and length of the fish. Various head, body, and caudal traits grew disproportionately faster in transgenic fish in both generations. The altered body shape of transgenic fish resulted in improved dressing percentage in the F2 generation. The carcass composition of transgenic muscle had a lower percentage of (P <0.01) moisture and lipids and higher (P <0.01) percentage of protein in both generations. Six of the 18 amino acids analyzed in F1 transgenic common carp muscle were higher F1 (P <0.05) than the control genotype; however, amino acid ratios were minimally changed. Also, the fatty acid profiles of both genotypes were minimally altered. Higher histidine and lysine ratios in the diet are recommended for maximum growth and health of transgenic common carp in intensive culture systems on the basis of essential amino acid ratios.     

Genomics - the new rock and roll?

The end of the beginning of the Human Genome Project was announced on 26 June when the working draft or first assembly was announced. Here, Ian Dunham who led the group at the Sanger Centre that produced the first complete sequence of a human chromosome reflects on how it felt to be with the genome project from the beginning.     

DNA sequence context modulates the impact of a cisplatin 1,2-d(GpG) intrastrand cross-link on the conformational and thermodynamic properties of duplex DNA

The anticancer activity of cisplatin derives from its ability to bind and cross-link DNA, with the major adduct being the 1,2-d(GpG) intrastrand cross-link. Here, the consequences of this adduct on the conformation, thermal stability, and energetics of duplex DNA are assessed, and the modulation of these parameters by the sequence context of the adduct is evaluated. The properties of a family of 15-mer DNA duplexes containing a single 1,2-d(GpG) cis-?Pt(NH(3))(2)?(2+) intrastrand cross-link are probed in different sequence contexts where the flanking base-pairs are systematically varied from T.A to C.G to A.T. By using a combination of spectroscopic and calorimetric techniques, the structural, thermal, and thermodynamic properties of each duplex, both with and without the cross-link, are characterized. Circular dichroism spectroscopic data reveal that the cross-link alters the structure of the host duplex in a manner consistent with a shift from a B-like to an A-like conformation. Thermal denaturation data reveal that the cross-link induces substantial thermal and thermodynamic destabilization of the host duplex. Significantly, the magnitudes of these cross-link-induced effects on duplex structure, thermal stability, and energetics are influenced by the bases that flank the adduct. The presence of flanking A.T base-pairs, relative to T.A or C.G base-pairs, enhances the extent of cross-link-induced alteration to an A-like conformation and dampens the extent of cross-link-induced duplex destabilization. These results are discussed in terms of available structural data, and in terms of the selective recognition of cisplatin-DNA adducts by HMG-domain proteins.     

Protein microarrays for highly parallel detection and quantitation of specific proteins and antibodies in complex solutions

Background

We describe a method for printing protein microarrays, and using these microarrays in a comparative fluorescence assay to measure the abundance of many specific proteins in complex solutions. A robotic device was used to print hundreds of specific antibody or antigen solutions in an array on the surface of derivatized microscope slides. Two complex protein samples, one serving as a standard for comparative quantitation, and the other representing an experimental sample in which the concentrations of specific proteins were to be measured, were labeled by covalent attachment of spectrally-resolvable fluorescent dyes. Specific antibody-antigen interactions localized specific components of the complex mixtures to defined cognate spots in the array, where the relative intensity of the fluorescent signals representing the experimental sample and the reference standard provided a measure of each protein's abundance in the experimental sample. To characterize the specificity, sensitivity and accuracy of this assay, we analyzed the performance of 115 antibody/antigen pairs.

Results

50% of the arrayed antigens, and 20% of the arrayed antibodies, provided specific and accurate measurements of their cognate ligands at or below concentrations of 1.6 µg/ml and 0.34 µg/ml, respectively. Some of the antibody/antigen pairs allowed detection of the cognate ligands at absolute concentrations below 1 ng/ml, and partial concentrations of less than 1 part in 10⁶, sensitivities sufficient for measurement of many clinically important proteins in patient blood samples.

Conclusions

Protein microarrays can provide a simple and practical means to characterize patterns of variation in hundreds or thousands of different proteins, in clinical or research applications.