Identification and characterisation of a human calmodulin-stimulated phosphodiesterase PDE1B1

A cDNA encoding a calmodulin-stimulated 3',5'-cyclic nucleotide phosphodiesterase (PDE) was isolated from a human brain cDNA library. The cDNA, designated HSPDE1B1, encoded a protein of 536 amino acids that shared 96% sequence identity with the bovine "63 kDa" calmodulin-stimulated PDE. The recombinant protein had cyclic nucleotide phosphodiesterase activity that was stimulated approximately 2-fold by Ca2+/calmodulin and preferred cGMP as substrate. In addition, the enzymatic activity of HSPDE1B1 was inhibited by phosphodiesterase inhibitors with potencies similar to that displayed toward the bovine PDE1 enzymes: IBMX approximately equal to 8-methoxymethyl-IBMX > vinpocetine approximately equal to zaprinast > cilostamide > rolipram. HSPDE1B1 mRNA was found predominantly in the brain. Lower mRNA levels were found in heart and skeletal muscle. In situ hybridisation of brain revealed expression of HSPDE1B1 predominately in neuronal cells of the cerebellum, hippocampus and caudate. The HSPDE1B1 gene was mapped to human chromosome 12. A partial genomic sequence of HSPDE1B1 was isolated and shown to contain two splice junctions that are conserved in the rat PDE4 and the Drosophila dunce genes.     

Proteoglycan expression by human trabecular meshworks

The aim of the present study was to clinically assess the peri-implant and periodontal conditions 1 year after placement of oral implants (ITI Dental Implant System) in partially edentulous patients. In all, 127 patients (median age 50 years, range 17 to 79) were examined. They were all treated according to a concept of comprehensive dental care and had received fixed partial dentures (FPD). Significant differences were observed between implants and contralateral control teeth with respect to mean pocket probing depth (PPD) (2.55 mm at implants/2.02 mm at teeth), mean probing attachment level (PAL) (2.97 mm/2.53 mm) and bleeding on probing (BOP) (24%/12%) (Wilcoxon matched pairs sign rank test, P < or = 0.01), whereas mean modified plaque index (0.22/0.30), mean modified bleeding index (0.35/0.44) and mean recession (-0.42 mm/-0.51 mm) did not significantly differ between implants and teeth. Compared to control teeth, the width of keratinized mucosa at implants was significantly smaller at lingual, but not at buccal aspects. Regression analyses showed no significant association between the amount of keratinized mucosa and degree of inflammation. Recession, PPD and PAL were slightly influenced by the amount of keratinized mucosa indicating greater resistance to probing. Grouping the implants according to various lengths, type of fixation of the FPD or combination with natural teeth did not result in statistically significant different clinical parameters, whereas grouping according to different localization within the oral cavity did. For example, the mean PAL in 83 anterior implants was 2.52 mm, whereas 175 posterior implants had a mean PAL of 3.18 mm (Mann-Whitney U-test, P < or = 0.01). Regression analyses between the mean PAL for all implants in each patient and the mean PAL of the corresponding dentition revealed an r2 of 0.23 (P < or = 0.01). Using multiple regression analysis, the mean PAL of the implants showed to be significantly influenced by the combined factors "fullmouth" PII, "fullmouth" BOP and mean PAL of all teeth. The results of this study suggest that in partially edentulous patients the overall periodontal condition may influence the clinical condition around implants and thus reinforces the importance of periodontal treatment prior to and supportive periodontal therapy after the incorporation of osseointegrated oral implants.     

Birthdate and cell marker analysis of scrambler: a novel mutation affecting cortical development with a reeler-like phenotype

The reeler mutation in mice produces an especially well characterized disorder, with systematically abnormal migration of cerebral cortical neurons. The reeler gene encodes a large protein, termed Reelin, that in the cortex is synthesized and secreted exclusively in the Cajal-Retzius neurons of the cortical marginal zone (D'Arcangelo et al., 1995). In reeler mutant mice, loss of Reelin protein is associated with a systematic loss of the normal, "inside-out" sequence of neurogenesis in the cortex: neurons are formed in the normal sequence but become localized in the cortex in a somewhat inverted, although relatively disorganized "outside-in" pattern. Here we show that the scrambler mutant mouse exhibits a loss of lamination in the cortex and hippocampus that is indistinguishable from that seen in the reeler mouse. We use BrdU birthdating studies to show that scrambler cortex shows a somewhat inverted "outside-in" sequence of birthdates for cortical neurons that is similar to that previously described in reeler cortex. Finally, we perform staining with the CR-50 monoclonal antibody (Ogawa et al., 1995), which recognizes the Reelin protein (D'Arcangelo et al., 1997). We show that Reelin immunoreactivity is present in the scrambler cortex in a normal pattern, suggesting that Reelin is synthesized and released normally. Our data suggest that scrambler is a mutation in the same gene pathway as the reeler gene (Relnrl) and is most likely downstream of Relnrl.     

Reasons adolescents don't use drugs: exploring a "depth of acceptance" model

Adolescent's reasons for not using drugs were examined for evidence of factors that might lead to differential resistance to drug use. Six thousand eight-hundred and forty-one sixth, eighth, tenth, and twelfth grade students were administered a comprehensive drug survey which included: 1) demographic information, 2) reasons not to use drugs, and 3) self-reports of lifetime and current (30-day) drug use across sixteen drug categories. The reasons for not using drugs were then factor analyzed and the results compared to a "Depth of Acceptance" (DOA) model consisting of four orientations: External Outcome, Social, and Personal. These orientations are thought both to represent distinct immunization factors and to be differentially related in strength to lifetime and current drug use. Confirmatory factor analysis revealed a close fit between the hypothesized orientations and factor loadings among the sixteen items. Collectively, the sixteen items were also found to be excellent predictors of both lifetime and current drug use. While multiple stepwise regression analyses did reveal differential predictive strengths between orientation and drug use, the misclassification of a single item apparently attenuated the results for Social Orientation. The DOA model appears to provide a useful framework for "fine-tuning" prevention messages based on factors that immunize against drug use.     

Coordination of lip muscle activity by 2-year-old children during speech and nonspeech tasks

This investigation was designed to quantify the coordinative organization of lip muscle activity of 2-year-old children during speech and nonspeech behaviors. Electromyographic (EMG) recordings of right upper and lower lip activity of seven 2-year-old children were obtained during productions of chewing, syllable repetition, lip protrusion, and speech (repeated two-word utterances) tasks. Task comparisons revealed that the coordinative organization of upper and lower lip activity is task specific; different coordinative strategies are employed for different tasks. Lip protrusion and syllable repetition tasks yielded strong coupling of upper and lower lip activity. Lip rounding (sentences containing the lip-rounding vowel /u/) and "nonlabial" speech tasks (sentences free of bilabials and lip-rounding vowels) resulted in low coupling of upper and lower lip activity. Moderate levels of coupling of upper and lower lip activity were evident for chewing and bilabial speech tasks (sentences loaded with bilabial plosion). This finding, that the coordinative elements of the perioral system of 2-year-olds are task specific, extends the results of previous studies of adults and children, where task-specific coordinative strategies were employed by the mandibular and perioral systems (Moore, 1993; Moore & Ruark, 1996; Moore, Smith, & Ringel, 1988; Wohlert & Goffman, 1994). The task-dependent coordination of the perioral system of 2-year-olds supports the notion that developing speech and earlier developing oromotor behaviors (i.e., sucking, chewing) are mediated by different control mechanisms.     

Selective peptidic and peptidomimetic inhibitors of Candida albicans myristoylCoA: protein N-myristoyltransferase: a new approach to antifungal therapy

MyristoylCoA: protein N-myristoyltransferase (NMT) catalyzes the cotranslational covalent attachment of a rare cellular fatty acid, myristate, to the N-terminal Gly residue of a variety of eukaryotic proteins. The myristoyl moiety is often essential for expression of the biological functions for these proteins. Attachment of C14:0 alone provides barely enough hydrophobicity to allow stable association with membranes. The partitioning of N-myrisotylproteins is therefore often modulated by "switches" that function through additional covalent or noncovalent modifications. Candida albicans, the principal cause of systemic fungal infection in immunocompromised humans, contains a single NMT gene that is essential for its viability. The functional properties of the acylCoA binding site of human and C. albicans NMT are very similar. However, there are distinct differences in their peptide binding sites. An ADP ribosylation factor (Arf) is included among the few cellular protein substrates of the fungal enzyme. Alanine scanning mutagenesis of an octapeptide derived from an N-terminal Arf sequence (GLYASKLS-NH2) disclosed that Gly1, Ser5, and Lys6 play predominant roles in binding. ALYASKLS-NH2 is an inhibitor competitive for peptide [Ki(app) = 15.3 +/- 6.4 microM] and noncompetitive for myristoylCoA. Remarkably, replacement of the N-terminal tetrapeptide with an 11-aminoundecanoyl group results in a competitive inhibitor (11-aminoundecanoyl-SKLS-NH2) that is approximately 40-fold more potent [Ki(app) = 0.40 +/- 0.03 microM] than the starting octapeptide. Removal of Leu-Ser from the C-terminus generates a competitive dipeptide inhibitor (11-aminoundecanoyl-SK-NH2) with a Ki(app) of 11.7 +/- 0.4 microM, equivalent to that of the starting octapeptide. A derivative dipeptide inhibitor containing a C-terminal N-cyclohexylethyl lysinamide moiety has the advantage of being more potent (IC50 = 0.11 +/- 0.03 microM) and resistant to digestion by cellular carboxypeptidases. Rigidifying the flexible aminoundecanoyl chain results in very potent general NMT inhibitors (IC50 = 40-50 nM). Substituting a 2-methylimidazole for the N-terminal amine and adding a benzylic alpha-methyl group with R stereochemistry to the rigidifying element produces even more potent inhibitors (IC50 = 20-50 nM) that are up to 500-fold selective for the fungal compared to human enzyme. A related less potent member of this series of compounds is fungistatic. Its growth inhibitory effects are associated with a reduction in cellular protein N-myristoylation, judged using cellular Arf as a reporter. These studies establish that NMT is a new antifungal target.     

An endogenous calcium oscillator may control early embryonic division

Transient elevations in the concentration of free cytosolic calcium ion ([Ca2+]i) promote cell phase transitions in early embryonic division and persist even if these transitions are blocked. These observations suggest that a [Ca2+]i oscillator is an essential timing element of the early embryonic "master clock." We explore this possibility by coupling a [Ca2+]i oscillator model to an early embryonic cell cycle model based on the protein interactions that govern the activity of the M-phase-promoting factor (MPF). We hypothesize three dynamical states of the MPF system and choose parameter sets to represent each. We then investigate how [Ca2+]i dynamics may control early embryonic division in both sea urchin and Xenopus embryos. To investigate both systems, distinct [Ca2+]i profiles matching those observed in sea urchin embryos (in which [Ca2+]i exhibits sharp transients) and Xenopus embryos (in which [Ca2+]i is elevated and oscillates sinusoidally) are imposed on each of the hypothesized dynamical states of MPF. In the first hypothesis, [Ca2+]i oscillations entrain the autonomous MPF oscillator. In the second and third hypotheses, where the MPF system rests in excitatory and bistable states, respectively, [Ca2+]i oscillations drive MPF activation cycles. Simulation results show that hypotheses two and three, in which a [Ca2+]i oscillator is a fundamental timing element of the master clock, best account for key experimental observations and the questions that they raise. Finally, we propose experiments to elucidate further [Ca2+]i regulation and the fundamental components of the early embryonic master clock.     

A Murine Oral‐Exposure Model for Nano‐ and Micro‐Particulates: Demonstrating Human Relevance with Food‐Grade Titanium Dioxide

Human exposure to persistent, nonbiological nanoparticles and microparticles via the oral route is continuous and large scale (10¹²–10¹³ particles per day per adult in Europe). Whether this matters or not is unknown but confirmed health risks with airborne particle exposure warns against complacency. Murine models of oral exposure will help to identify risk but, to date, lack validation or relevance to humans. This work addresses that gap. It reports i) on a murine diet, modified with differing concentrations of the common dietary particle, food grade titanium dioxide (fgTiO₂), an additive of polydisperse form that contains micro‐ and nano‐particles, ii) that these diets deliver particles to basal cells of intestinal lymphoid follicles, exactly as is reported as a “normal occurrence” in humans, iii) that confocal reflectance microscopy is the method of analytical choice to determine this, and iv) that food intake, weight gain, and Peyer's patch immune cell profiles, up to 18 weeks of feeding, do not differ between fgTiO₂‐fed groups or controls. These findings afford a human‐relevant and validated oral dosing protocol for fgTiO₂ risk assessment as well as provide a generalized platform for application to oral exposure studies with nano‐ and micro‐particles.     

Clinical and genetic analysis of primary bilateral adrenal diseases (micro- and macronodular disease) leading to Cushing syndrome

Primary bilateral adrenocortical diseases are rare entities that have recently been appreciated as potential causes of Cushing syndrome. They include (i) primary pigmented adrenocortical disease (PPNAD), also known as "micronodular adrenal disease", which is a genetic disorder that is often associated with Carney complex, and (ii) massive macronodular adrenocortical disease (MMAD), a rare disorder of unknown etiology that affects older adults. Carney complex is a multiple endocrine neoplasia (MEN) syndrome that affects not only the adrenal cortex, but also the pituitary, thyroid, and gonads. It is associated with pigmentation abnormalities as well as myxomas and other mesenchymal and neural crest neoplasms. The inheritance of the complex is autosomal dominant, and genetic mapping has shown that at least two loci are involved in its pathogenesis. MMAD appears to be an isolated finding in most cases, and a genetic defect has not yet been defined. Ectopic expression of hormone receptors has been implicated in several cases of MMAD, but an underlying deficit has not been detected. Bilateral adrenocortical hyperplasia has also been described in McCune-Albright syndrome and MEN type-1, but this finding is not always associated with hypercortisolism. The genetic defects for these diseases are known, but their role in adrenal cortex pathophysiology has not been fully elucidated. Identification of the molecular defects responsible for bilateral adrenocortical disorders is expected to shed light on many aspects of early adrenal gland differentiation and tumorigenesis.