A mouse model expressing a truncated form of ameloblastin exhibits dental and junctional epithelium defects

Ameloblastin (AMBN) is the second most abundant extracellular matrix protein produced by the epithelial cells called ameloblasts and is found mainly in forming dental enamel. Inactivation of its expression by gene knockout results in absence of the enamel layer and its replacement by a thin layer of dysplastic mineralized matrix. The objective of this study was to further characterize the enamel organ and mineralized matrix produced in the AMBN knockout mouse. However, in the course of our study, we unexpectedly found that this mouse is in fact a mutant that does not express the full-length protein but that produces a truncated form of AMBN. Mandibles from wild type and mutant mice were processed for morphological analyses and immunolabeling. Microdissected enamel organs and associated matrix were also prepared for molecular and biochemical analyses. In incisors from mutants, ameloblasts lost their polarized organization and the enamel organ detached from the tooth surface and became disorganized. A thin layer of dysplastic mineralized material was deposited onto dentin, and mineralized masses were present within the enamel organ. These mineralized materials generated lower backscattered electron contrast than normal enamel, and immunocytochemistry with colloidal gold revealed the presence of amelogenin, bone sialoprotein and osteopontin. In addition, the height of the alveolar bone was reduced, and the junctional epithelium lost its integrity. Immunochemical and RT–PCR results revealed that the altered enamel organ in the mutant mice produced a shorter AMBN protein that is translated from truncated RNA missing exons 5 and 6. These results indicate that absence of full-length protein and/or expression of an incomplete protein have direct/indirect effects beyond structuring of mineral during enamel formation, and highlight potential functional regions on the AMBN molecule.     

ACROSS‐ENVIRONMENT GENETIC CORRELATIONS AND THE FREQUENCY OF SELECTIVE ENVIRONMENTS SHAPE THE EVOLUTIONARY DYNAMICS OF GROWTH RATE IN IMPATIENS CAPENSIS

Trade‐offs can exist within and across environments, and constrain evolutionary trajectories. To examine the effects of competition and resource availability on trade‐offs, we grew individuals of recombinant inbred lines of Impatiens capensis in a factorial combination of five densities with two light environments (full light and neutral shade) and used a Bayesian logistic growth analysis to estimate intrinsic growth rates. To estimate across‐environment constraints, we developed a variance decomposition approach to principal components analysis, which accounted for sample size, model‐fitting, and within‐RIL variation prior to eigenanalysis. We detected negative across‐environment genetic covariances in intrinsic growth rates, although only under full‐light. To evaluate the potential importance of these covariances, we surveyed natural populations of I. capensis to measure the frequency of different density environments across space and time. We combined our empirical estimates of across‐environment genetic variance–covariance matrices and frequency of selective environments with hypothetical (yet realistic) selection gradients to project evolutionary responses in multiple density environments. Selection in common environments can lead to correlated responses to selection in rare environments that oppose and counteract direct selection in those rare environments. Our results highlight the importance of considering both the frequency of selective environments and the across‐environment genetic covariances in traits simultaneously.     

Synthesis and antioxidant properties of pulvinic acids analogues

The synthesis of three types of pulvinic acid analogues, using a diversity-oriented strategy starting from a single compound, dimethyl l-tartrate, is described. Lacey-Dieckmann condensation, alcohol dehydration and Suzuki-Miyaura cross-couplings were employed in the course of the analogues syntheses. The evaluation of the antioxidant properties of the 28 synthesized analogues was carried out using antioxidant capacity assays (protection of thymidine and β-carotene) and free radical scavenging assays (DPPH radical and ABTS radical cation). This allowed to assess the relative influence of the groups bonded to the tetronic ring and to the exocyclic double bond on the activity, as well as the importance of this exocyclic double bond. It was shown that the presence of an electron-donating group on the 3-position of the tetronic ring had a beneficial effect. It was shown in several assays that the presence of the exocyclic bond was not crucial to the activity.     

Start-up and stabilization of an Anammox process from a non-acclimatized sludge in CSTR

Development of an Anammox (anaerobic ammonium oxidation) process using non-acclimatized sludge requires a long start-up period owing to the very slow growth rate of Anammox bacteria. This article addresses the issue of achieving a shorter start-up period for Anammox activity in a well-mixed continuously stirred tank reactor (CSTR) using non-acclimatized anaerobic sludge. Proper selection of enrichment conditions and low stirring speed of 30 ± 5 rpm resulted in a shorter start-up period (82 days). Activity tests revealed the microbial community structure of Anammox micro-granules. Ammonia-oxidizing bacteria (AOB) were found on the surface and on the outer most layers of granules while nitrite-oxidizing bacteria (NOB) and Anammox bacteria were present inside. Fine-tuning of influent NO₂ ⁻/NH₄ ⁺ ratio allowed Anammox activity to be maintained when mixed microbial populations were present. The maximum nitrogen removal rate achieved in the system was 0.216 kg N/(m³ day) with a maximum specific nitrogen removal rate of 0.434 g N/(g VSS day). During the study period, Anammox activity was not inhibited by pH changes and free ammonia toxicity.     

Quantification of acetylcholine, choline, betaine, and dimethylglycine in human plasma and urine using stable-isotope dilution ultra performance liquid chromatography-tandem mass spectrometry

Disorders in choline metabolism are related to disease conditions. We developed a stable-isotope dilution ultra performance liquid chromatography-mass spectrometry (UPLC-MS/MS) method for the simultaneous quantification of acetylcholine (ACh), betaine, choline, and dimethylglycine (DMG). We used this method to measure concentrations of the analytes in plasma and urine in addition to other biological fluids after a protein precipitation by acetonitrile. The detection limits were between 0.35 nmol/L (for ACh in urine) and 0.34 μmol/L (for betaine in urine). ACh concentrations were not detectable in plasma. Intraassay and interassay coefficient of variation (CVs) were all <10.0% in biological fluids, except for DMG in cerebrospinal fluid (CV=12.44%). Mean recoveries in urine pool samples were between 99.2% and 103.9%. The urinary excretion of betaine, choline, and DMG was low, with approximately 50.0% higher excretion of choline in females compared to males. Median urinary excretion of ACh were 3.44 and 3.92 μmol/mol creatinine in males and females, respectively (p=0.689). Plasma betaine concentrations correlated significantly with urinary excretions of betaine (r=0.495, p=0.027) and choline (r=0.502, p=0.024) in females. Plasma choline concentrations correlated significantly with urinary excretion of ACh in males (r=0.419, p=0.041) and females (r=0.621, p=0.003). The new method for the simultaneous determination of ACh, betaine, choline, and DMG is sensitive, precise, and fast enough to be used in clinical investigations related to the methylation pathway.     

Stable isotope-resolved metabolomic analysis of lithium effects on glial-neuronal metabolism and interactions

Despite the long-established therapeutic efficacy of lithium in the treatment of bipolar disorder (BPD), its molecular mechanism of action remains elusive. Newly developed stable isotope-resolved metabolomics (SIRM) is a powerful approach that can be used to elucidate systematically how lithium impacts glial and neuronal metabolic pathways and activities, leading ultimately to deciphering its molecular mechanism of action. The effect of lithium on the metabolism of three different ¹³C-labeled precursors ([U-¹³C]-glucose, ¹³C-3-lactate or ¹³C-2,3-alanine) was analyzed in cultured rat astrocytes and neurons by nuclear magnetic resonance (NMR) spectroscopy and gas chromatography mass spectrometry (GC-MS). Using [U-¹³C]-glucose, lithium was shown to enhance glycolytic activity and part of the Krebs cycle activity in both astrocytes and neurons, particularly the anaplerotic pyruvate carboxylation (PC). The PC pathway was previously thought to be active in astrocytes but absent in neurons. Lithium also stimulated the extracellular release of ¹³C labeled-lactate, -alanine (Ala), -citrate, and -glutamine (Gln) by astrocytes. Interrogation of neuronal pathways using ¹³C-3-lactate or ¹³C-2,3-Ala as tracers indicated a high capacity of neurons to utilize lactate and Ala in the Krebs cycle, particularly in the production of labeled Asp and Glu via PC and normal cycle activity. Prolonged lithium treatment enhanced lactate metabolism via PC but inhibited lactate oxidation via the normal Krebs cycle in neurons. Such lithium modulation of glycolytic, PC and Krebs cycle activity in astrocytes and neurons as well as release of fuel substrates by astrocytes should help replenish Krebs cycle substrates for Glu synthesis while meeting neuronal demands for energy. Further investigations into the molecular regulation of these metabolic traits should provide new insights into the pathophysiology of mood disorders and early diagnostic markers, as well as new target(s) for effective therapies.     

Cloning and analysis of Nkx6.3 during CNS and gastrointestinal development

Members of the Nkx family of homeodomain proteins are involved in a variety of developmental processes such as cell fate determination in the CNS and in the pancreas. Here we describe the cloning and developmental expression pattern of Nkx6.3, a new member of the Nkx6 subfamily of homeodomain proteins. Nkx6.3 is expressed in the developing CNS and gastro-intestinal tract. In contrast to Nkx6.1 and Nkx6.2 that are broadly expressed in ventral positions of the developing CNS, Nkx6.3 shows a remarkably selective expression in a subpopulation of differentiating V2 neurons at caudal hindbrain levels. The expression of Nkx6.3 at this level depends on the activity of other Nkx6 proteins. In the gut, Nkx6.3 is expressed in duodenal and glandular stomach endoderm and at the end of gestation Nkx6.3 became restricted to the base of the gastric units in the glandular stomach. The expression of Nkx6.3 overlapped with the expression of Nkx6.2 both in the CNS and in the gut. Transient Nkx6.2 expression was also detected in the developing pancreas. However, analysis of Nkx6.2(-/-) mice did not display any obvious aberrations of pancreatic or stomach development.     

Infectious etiology in multiple sclerosis: the debate continues

‘Demonstrating Infectious Cause: Viral and Bacterial Infections in MS and Related Disorders’ was held in Brighton, UK, 23–25 August 1999.     

Comparative immunogenicity in rhesus monkeys of DNA plasmid,recombinant vaccinia virus,and replication-defective adenovirus vectors expressing a human immunodeficiency virus type 1 gag gene

Cellular immune responses, particularly those associated with CD3⁺ CD8⁺ cytotoxic T lymphocytes (CTL), play a primary role in controlling viral infection, including persistent infection with human immunodeficiency virus type 1 (HIV-1). Accordingly, recent HIV-1 vaccine research efforts have focused on establishing the optimal means of eliciting such antiviral CTL immune responses. We evaluated several DNA vaccine formulations, a modified vaccinia virus Ankara vector, and a replication-defective adenovirus serotype 5 (Ad5) vector, each expressing the same codon-optimized HIV-1 gag gene for immunogenicity in rhesus monkeys. The DNA vaccines were formulated with and without one of two chemical adjuvants (aluminum phosphate and CRL1005). The Ad5-gag vector was the most effective in eliciting anti-Gag CTL. The vaccine produced both CD4⁺ and CD8⁺ T-cell responses, with the latter consistently being the dominant component. To determine the effect of existing antiadenovirus immunity on Ad5-gag-induced immune responses, monkeys were exposed to adenovirus subtype 5 that did not encode antigen prior to immunization with Ad5-gag. The resulting anti-Gag T-cell responses were attenuated but not abolished. Regimens that involved priming with different DNA vaccine formulations followed by boosting with the adenovirus vector were also compared. Of the formulations tested, the DNA-CRL1005 vaccine primed T-cell responses most effectively and provided the best overall immune responses after boosting with Ad5-gag. These results are suggestive of an immunization strategy for humans that are centered on use of the adenovirus vector and in which existing adenovirus immunity may be overcome by combined immunization with adjuvanted DNA and adenovirus vector boosting.