Evidence for regulation of amelogenin gene expression by 1,25-dihydroxyvitamin D(3) in vivo

The unique hereditary enamel defect clearly related to the disturbance of one enamel matrix protein is X-linked amelogenesis imperfecta (AI), in which several mutations of amelogenin gene have been identified. The clinical phenotype of many of these subjects shows similarities with enamel defects related to rickets. Therefore, we hypothesized that rachitic dental dysplasia is related to disturbances in the amelogenin pathway. In order to test this hypothesis, combined qualitative and quantitative studies in experimental vitamin D-deficient (-D) rat model systems were performed. First, Western blot analysis of microdissected enamel matrix (secretion and maturation stages) showed no clear evidence of dysregulation of amelogenin protein processing in -D rats as compared with the controls. Second, the ultrastructural investigation permitted identification of the internal tissular defect of rachitic enamel, the irregular absence of intraprismatic enamel observed in -D animals, suggesting a possible link between prism morphogenesis and vitamin D. In addition, the steady-state levels of amelogenin mRNAs measured in microdissected dental cells was decreased in -D rats and up-regulated by an unique injection of 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)). The present study shows evidences that amelogenin expression is regulated by vitamin D. This is the first study of an hormonal regulation of tooth-specific genes.     

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.     

Global distribution of nearly identical phage-encoded DNA sequences

Phages, the most abundant biological entities on the planet, play important roles in biogeochemical cycling, horizontal gene transfer, and defining microbial community composition. However, very little is known about phage diversity or biogeography, and there has not yet been a systematic effort to compare the phages found in different ecosystems. Here, we report that T7-like Podophage DNA polymerase sequences occur in every major biome investigated, including marine, freshwater, sediment, terrestrial, extreme, and metazoan-associated. The majority of these sequences belong to a unique clade that is only distantly related to cultured isolates. Some identical T7-like phage-encoded DNA polymerase genes from this clade were >99% conserved at the nucleotide level in multiple different environments, suggesting that these phages are moving between biomes in recent evolutionary time and that the global genomic pool for T7-like phages may be smaller than previously hypothesized.     

Hydrogen/deuterium exchange studies of native rabbit MM-CK dynamics

Creatine kinase (CK) isoenzymes catalyse the reversible transfer of a phosphoryl group from ATP onto creatine. This reaction plays a very important role in the regulation of intracellular ATP concentrations in excitable tissues. CK isoenzymes are highly resistant to proteases in native conditions. To appreciate localized backbone dynamics, kinetics of amide hydrogen exchange with deuterium was measured by pulse-labeling the dimeric cytosolic muscle CK isoenzyme. Upon exchange, the protein was digested with pepsin, and the deuterium content of the resulting peptides was determined by liquid chromatography coupled to mass spectrometry (MS). The deuteration kinetics of 47 peptides identified by MS/MS and covering 96% of the CK backbone were analyzed. Four deuteration patterns have been recognized: The less deuterated peptides are located in the saddle-shaped core of CK, whereas most of the highly deuterated peptides are close to the surface and located around the entrance to the active site. Their exchange kinetics are discussed by comparison with the known secondary and tertiary structures of CK with the goal to reveal the conformational dynamics of the protein. Some of the observed dynamic motions may be linked to the conformational changes associated with substrate binding and catalytic mechanism.     

Phosphoproteome analysis of capacitated human sperm. Evidence of tyrosine phosphorylation of a kinase-anchoring protein 3 and valosin-containing protein/p97 during capacitation

Before fertilization can occur, mammalian sperm must undergo capacitation, a process that requires a cyclic AMP-dependent increase in tyrosine phosphorylation. To identify proteins phosphorylated during capacitation, two-dimensional gel analysis coupled to anti-phosphotyrosine immunoblots and tandem mass spectrometry (MS/MS) was performed. Among the protein targets, valosin-containing protein (VCP), a homolog of the SNARE-interacting protein NSF, and two members of the A kinase-anchoring protein (AKAP) family were found to be tyrosine phosphorylated during capacitation. In addition, immobilized metal affinity chromatography was used to investigate phosphorylation sites in whole protein digests from capacitated human sperm. To increase this chromatographic selectivity for phosphopeptides, acidic residues in peptide digests were converted to their respective methyl esters before affinity chromatography. More than 60 phosphorylated sequences were then mapped by MS/MS, including precise sites of tyrosine and serine phosphorylation of the sperm tail proteins AKAP-3 and AKAP-4. Moreover, differential isotopic labeling was developed to quantify phosphorylation changes occurring during capacitation. The phosphopeptide enrichment and quantification methodology coupled to MS/MS, described here for the first time, can be employed to map and compare phosphorylation sites involved in multiple cellular processes. Although we were unable to determine the exact site of phosphorylation of VCP, we did confirm, using a cross-immunoprecipitation approach, that this protein is tyrosine phosphorylated during capacitation. Immunolocalization of VCP showed fluorescent staining in the neck of noncapacitated sperm. However, after capacitation, staining in the neck decreased, and most of the sperm showed fluorescent staining in the anterior head.     

H-7 and fetal calf serum (FCS) act synergistically to increase apoptosis in the KB line of human oral carcinoma cells

There is a high incidence of oral squamous cell carcinoma (SCC) worldwide. The survival rate is among the lowest of the major cancers and has not improved significantly over the past two decades. The KB line of human oral carcinoma cells is a useful experimental system for studies of the biology of oral SCC. In a previous study, we reported inhibition of KB cell proliferation and stimulation of desmosome formation in confluent cultures treated with 20 microM H-7 (1-(5-isoquinolinylsulfonyl)-2-methylpiperazine). In the present study, the effects of this protein kinase C (PKC) inhibitor on the survival of KB cells were investigated. Apoptotic cells were detected using a combination of Hoechst 33258 nuclear stain, TUNEL technique and ultrastructural analysis. Our results indicated that H-7 significantly increased apoptosis in KB cells in a dose-dependent manner. Maximal stimulation occurred at 100 microM, the highest dose of H-7 tested. Apoptotic cells exhibited nuclear fragmentation, chromatin condensation and apoptotic bodies. Interestingly, H-7 and fetal calf serum (FCS) acted synergistically to increase apoptosis in KB cells, suggesting that there is a serum activated subpopulation of H-7 target cells in the cultures. The underlying mechanism of activation remains to be elucidated. Our study suggests that the PKC inhibitor H-7 is a potentially useful cytostatic agent for oral carcinoma cells.     

FastGroup: A program to dereplicate libraries of 16S rDNA sequences

Background

Ribosomal 16S DNA sequences are an essential tool for identifying and classifying microbes. High-throughput DNA sequencing now makes it economically possible to produce very large datasets of 16S rDNA sequences in short time periods, necessitating new computer tools for analyses. Here we describe FastGroup, a Java program designed to dereplicate libraries of 16S rDNA sequences. By dereplication we mean to: 1) compare all the sequences in a data set to each other, 2) group similar sequences together, and 3) output a representative sequence from each group. In this way, duplicate sequences are removed from a library.

Results

FastGroup was tested using a library of single-pass, bacterial 16S rDNA sequences cloned from coral-associated bacteria. We found that the optimal strategy for dereplicating these sequences was to: 1) trim ambiguous bases from the 5' end of the sequences and all sequence 3' of the conserved Bact517 site, 2) match the sequences from the 3' end, and 3) group sequences >=97% identical to each other.

Conclusions

The FastGroup program simplifies the dereplication of 16S rDNA sequence libraries and prepares the raw sequences for subsequent analyses.     

A mass spectrometry method for measuring 15N incorporation into pheophytin

The 15N content of pheophytin, the magnesium-free derivative of chlorophyll, can be measured with great accuracy and precision using positive-ion atmospheric pressure ionization electrospray mass spectroscopy following a simple solvent extraction of small amounts of plant tissue. The molecular weight of pheophytin prepared from Chlamydomonas reinhardtii grown in different ratios of 14N/15N showed linear regression with the isotopic input, with a precision of 0.5-1%. Using an isotope dilution strategy, we have shown that nitrogen fixation can contribute substantial 14N to pheophytin isolated from Medicago truncatula plants grown in symbiosis with Sinorhizobium meliloti. The assay is sensitive, precise, rapid, simple, and robust. These features suggest that it could become an important tool for measuring the contribution of symbiotic and associative nitrogen fixation to plant metabolism.     

Androgen receptor phosphorylation. Regulation and identification of the phosphorylation sites

Activation of signal transduction kinase cascades has been shown to alter androgen receptor (AR) activity. Although it has been suggested that changes in AR phosphorylation might be directly responsible, the basal and regulated phosphorylations of the AR have not been fully determined. We have identified the major sites of AR phosphorylation on ARs expressed in COS-1 cells using a combination of peptide mapping, Edman degradation, and mass spectrometry. We describe the identification of seven AR phosphorylation sites, show that the phosphopeptides seen with exogenously expressed ARs are highly similar to those seen with endogenous ARs in LNCaP cells and show that specific agonists differentially regulate the phosphorylation state of endogenous ARs in LNCaP prostate cancer cells. Treatment of LNCaP cells with the synthetic androgen, R1881, elevates phosphorylation of serines 16, 81, 256, 308, 424, and 650. Ser-94 appears constitutively phosphorylated. Forskolin, epidermal growth factor, and phorbol 12-myristate 13-acetate increase the phosphorylation of Ser-650. The kinetics of phosphorylation of most sites in response to hormone or forskolin is temporally delayed, reaching a maximum at 2 h post-stimulation. The exception is Ser-81, which continues to display increasing phosphorylation at 6 h. These data provide a basis for analyzing mechanisms of cross-talk between growth factor signaling and androgen in prostate development, physiology, and cancer.