The odontoblast process extends to the dentinoenamel junction: an immunocytochemical study of rat dentine

The length and extent of the odontoblast cell process in dentine has been the subject of controversy for many years. Here an immunofluorescence technique has been applied at the light microscope level to rat coronal dentine to localize the intracellular components actin and tubulin. Adult rats were perfused with periodate-lysine-paraformaldehyde fixative, teeth were extracted, the molar crowns were demineralized, dehydrated, wax embedded, and 6 micron sections were prepared. The sections were postfixed in -20 degrees C acetone and then incubated with affinity-purified rabbit anti-actin or anti-tubulin antibodies, followed by fluorescein-conjugated goat anti-rabbit immunoglobulin. Intratubular immunofluorescence labeling for tubulin extended to the dentinoenamel junction, whereas labeling for actin, although extending to the dentinoenamel junction, was more prominent in the pulpal third of the rat dentine. Areas in which odontoblast processes are known not to occur, i.e., the atubular dentine, were not labeled by either antibody. The presence of actin- and tubulin-containing structures extending to the dentinoenamel junction is consistent with the hypothesis that the odontoblast process traverses the dentine for up to 3-4 mm, all the way to the dentinoenamel junction. Furthermore, the different staining patterns for actin-containing microfilaments as compared to tubulin-containing microtubules suggest that these two filamentous systems may have different roles in the function of the odontoblast process.     

Structure and chromosome localization of the human eosinophil-derived neurotoxin and eosinophil cationic protein genes: Evidence for intronless coding sequences in the ribonuclease gene superfamily

Human genomic DNAs for the eosinophil granule proteins, eosinophil-derived neurotoxin (EDN) and eosinophil cationic protein (ECP), were isolated from genomic libraries. Alignment of EDN (RNS2) and ECP (RNS3) gene sequences demonstrated remarkable nucleotide similarities in noncoding sequences, introns, and flanking regions, as well as in the previously known coding regions. Detailed examination of the 5'-noncoding regions yielded putative TATA and CAAT boxes, as well as similarities to promoter motifs from unrelated genes. A single intron of 230 bases was found in the 5' untranslated region and we suggest that a single intron in this region and an intronless coding region are features common to many members of the RNase gene superfamily. The RNS2 and RNS3 genes were localized to the q24-q31 region of human chromosome 14. It is likely that these two genes arose as a consequence of a gene duplication event that took place approximately 25-40 million years ago and that a subset of anthropoid primates possess both of these genes or closely related genes.     

Multiple members of the UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferase family are essential for viability in Drosophila

Mucin-type O-glycosylation represents a major form of post-translational modification that is conserved across most eukaryotic species. This type of glycosylation is initiated by a family of enzymes (GalNAc-Ts in mammals and PGANTs in Drosophila) whose members are expressed in distinct spatial and temporal patterns during development. Previous work from our group demonstrated that one member of this family is essential for viability and another member modulates extracellular matrix composition and integrin-mediated cell adhesion during development. To investigate whether other members of this family are essential, we employed RNA interference (RNAi) to each gene in vivo. Using this approach, we identified 4 additional pgant genes that are required for viability. Ubiquitous RNAi to pgant4, pgant5, pgant7, or the putative glycosyltransferase CG30463 resulted in lethality. Tissue-specific RNAi was also used to define the specific organ systems and tissues in which each essential family member is required. Interestingly, each essential pgant had a unique complement of tissues in which it was required. Additionally, certain tissues (mesoderm, digestive system, and tracheal system) required more than one pgant, suggesting unique functions for specific enzymes in these tissues. Expanding upon our RNAi results, we found that conventional mutations in pgant5 resulted in lethality and specific defects in specialized cells of the digestive tract, resulting in loss of proper digestive system acidification. In summary, our results highlight essential roles for O-glycosylation and specific members of the pgant family in many aspects of development and organogenesis.     

Methylation of Smad6 by protein arginine N-methyltransferase 1

Signal transduction pathways utilize posttranslational modifications to regulate the activity of their components in a temporal-spatial and efficient fashion. Arginine methylation is one of the posttranslational modifications that can result in monomethylated-, asymmetric dimethylated- and/or symmetric dimethylated-arginine residues in proteins. Here we demonstrate that inhibitory-Smads (Smad6 and Smad7), but not receptor-regulated- (R-)Smads and the common-partner Smad4, can be methylated by protein arginine N-methyltransferase (PRMT)1. Using mass-spectrometric analysis, we found that PRMT1 dimethylates arginine(74) (Arg(74)) in mouse Smad6. PRMT1 interacts with the N-terminal domain of Smad6 in which Arg(74) residue is located. Assays examined so far have shown no significant differences between the functions of Smad6 and those of methylation-defective Smad6 (Smad6R74A). Both wild-type and Smad6R74A were equally efficient in blocking BMP-induced growth arrest upon their ectopic expression in HS-72 mouse B-cell hybridoma cells.     

Structural Nested Mean Models for Assessing Time-Varying Effect Moderation

Summary .  This article considers the problem of assessing causal effect moderation in longitudinal settings in which treatment (or exposure) is time varying and so are the covariates said to moderate its effect.  Intermediate causal effects  that describe time-varying causal effects of treatment conditional on past covariate history are introduced and considered as part of Robins' structural nested mean model. Two estimators of the intermediate causal effects, and their standard errors, are presented and discussed: The first is a proposed two-stage regression estimator. The second is Robins' G-estimator. The results of a small simulation study that begins to shed light on the small versus large sample performance of the estimators, and on the bias–variance trade-off between the two estimators are presented. The methodology is illustrated using longitudinal data from a depression study.     

Altered Receptor Specificity and Cell Tropism of D222G Hemagglutinin Mutants Isolated from Fatal Cases of Pandemic A(H1N1) 2009 Influenza Virus

Mutations in the receptor-binding site of the hemagglutinin of pandemic influenza A(H1N1) 2009 viruses have been detected sporadically. An Asp222Gly (D222G) substitution has been associated with severe or fatal disease. Here we show that 222G variants infected a higher proportion of ciliated cells in cultures of human airway epithelium than did viruses with 222D or 222E, which targeted mainly nonciliated cells. Carbohydrate microarray analyses showed that 222G variants bind a broader range of α2-3-linked sialyl receptor sequences of a type expressed on ciliated bronchial epithelial cells and on epithelia within the lung. These features of 222G mutants may contribute to exacerbation of disease.Although the majority of disease cases have been mild, the pandemic influenza A(H1N1) 2009 (H1N1pdm) virus has caused a substantial number of severe and fatal infections (2). Mutants with a D222G or D222E substitution (D225G or D225E in the H3 numbering system) in the receptor-binding site of the virus hemagglutinin (HA) have been detected sporadically (1), and the D222G substitution has been observed to correlate with cases of severe or fatal disease (1, 3, 9, 14). Cell surface receptors for influenza viruses are sialyl glycans (α2-3 Sia or α2-6 Sia) with terminal sialic acid linked α2-3 or α2-6, respectively, to a penultimate galactose. These differ in distribution in the tissues and cells of different species. The sialyl glycans are differentially recognized by the HAs of human and animal influenza viruses and are critical determinants of host range and tissue tropism (16). Using an experimental system of differentiated cultures of human tracheobronchial epithelial cells (HTBE) for studying influenza virus cell tropism, we and others have established that in the initial stages of infection, seasonal human influenza viruses which recognize α2-6 Sia receptors infect mainly nonciliated cells, whereas avian viruses which recognize α2-3 Sia receptors predominantly infect ciliated cells (8, 17, 22).Previous analyses of human and swine influenza H1N1 viruses (5, 15, 21) and preliminary studies of H1N1pdm viruses (24) have indicated that amino acid substitutions in the HA at position 222 may affect the specificity of receptor binding. This, in turn, would be predicted to determine the range of cell types in human respiratory tissues infected by the viruses (17, 20, 22, 23). We have therefore examined the influence of the D222G and D222E substitutions on the cell tropism of H1N1pdm viruses in HTBE cultures (Table ​(Table1).1). Five viruses were isolated from clinical material in MDCK cells and passaged solely in these cells. Two of these, A/Hamburg/5/2009 (Ham) (4) isolated from a case of mild infection and A/Moldova/G186/2009 (Mol) from a serious but nonfatal infection, had 222D. A/Dakar/37/2009 (Dak) isolated from a mild case of the disease had 222E. Two isolates from fatal cases, A/Lviv/N6/2009 (Lvi) and A/Norway/3206-3/2009 (Nor), had 222G. A sixth virus tested, A/Hamburg/5/2009-e (Ham-e), was derived from Ham by egg passage and plaque purification in MDCK cells and differed by a single substitution, D222G.

TABLE 1.

Differences in amino acid sequence of the HAs of the H1N1pdm viruses and cell tropism in HTBE cultures

Functional proteomic and structural insights into molecular targets related to the growth inhibitory effect of tanshinone IIA on HeLa cells

Certain antitumor agents have recently been extracted from the roots of Salvia miltiorrhiza Bunge. The diterpene derivative, tanshinone IIA, possesses cytotoxic activity against several human carcinoma cell lines. It also inhibits invasion and metastasis of cancer cells. In the present study, we isolated tanshinone IIA from S. miltiorrhiza, and it exhibited strong growth inhibition against human cervical cancer cells in dose‐ and time‐dependent manners with a 50% cell growth inhibition value of 2.5 μg/mL (8.49 μM). Flow cytometric analysis of cell cycle progression revealed that G₂/M arrest was initiated after a 24 h exposure to the drug. It also resulted in DNA fragmentation and degradation of poly (ADP‐ribose) polymerase indicating that tanshinone IIA may be a potential antitumor agent. Furthermore, we performed a comprehensive proteomic analysis to survey global protein changes induced by tanshinone IIA treatment on HeLa cells. Significant changes in the levels of cytoskeleton proteins as well as stress‐associated proteins were observed. Immunoblot analysis and immunofluorescence staining were used to confirm the levels of protein expression. Overexpression of the vimentin rescued these tanshinone IIA‐induced events. Computational docking methods indicated that tanshinone IIA could stably bind to the β‐subunit of the microtubule protein. An interaction network analysis of these 12 proteins using MetaCore? software suggested that tanshinone IIA treatment regulated the expressions of proteins involved in apoptotic processes, spindle assembly, and p53 activation, including vimentin, Maspin, α‐ and β‐tubulin, and GRP75. Taken together, our results suggest that tanshinone IIA strongly inhibited the growth of cervical cancer cells through interfering in the process of microtubule assembly, leading to G₂/M phase arrest and sequent apoptosis. The success of this large‐scale effort was assessed by a bioinformatics analysis of proteins through predictions of protein domains and possible functional roles. The possible contributions of these proteins to the cytotoxicity of tanshinone IIA provide potential opportunities for the development of cancer therapeutics.