Permissive Conformations of a Transmembrane Helix Allow Intramembrane Proteolysis by γ-Secretase

The intramembrane protease γ-secretase activates important signaling molecules, such as Notch receptors. It is still unclear, however, how different elements within the primary structure of substrate transmembrane domains (TMDs) contribute to their cleavability. Using a newly developed yeast-based cleavage assay, we identified three crucial regions within the TMDs of the paralogs Notch1 and Notch3 by mutational and gain-of-function approaches. The AAAA or AGAV motifs within the N-terminal half of the TMDs were found to confer strong conformational flexibility to these TMD helices, as determined by mutagenesis coupled to deuterium/hydrogen exchange. Crucial amino acids within the C-terminal half may support substrate docking into the catalytic cleft of presenilin, the enzymatic subunit of γ-secretase. Further, residues close to the C-termini of the TMDs may stabilize a tripartite β-sheet in the substrate/enzyme complex. NMR structures reveal different extents of helix bending as well as an ability to adopt widely differing conformational substates, depending on the sequence of the N-terminal half. The difference in cleavability between Notch1 and Notch3 TMDs is jointly determined by the conformational repertoires of the TMD helices and the sequences of the C-terminal half, as suggested by mutagenesis and building molecular models. In sum, cleavability of a γ-secretase substrate is enabled by different functions of cooperating TMD regions, which deepens our mechanistic understanding of substrate/non-substrate discrimination in intramembrane proteolysis.     

Structure and composition of the incisor enamel of extant and fossil mammals with tooth pigmentation

The inclusion of iron compounds in teeth, which impart a red to orange colour to them, is a phenomenon shown by several groups of vertebrates in different periods of their evolution. Incisors from fossil and extant shrews and from extant rodents were sectioned and studied with the techniques of scanning electron microscopy (SEM), transmission electron microscopy (TEM) and scanning transmission electron microscopy (STEM) to compare their structure and the distribution of Fe. The enamel in white‐ and red‐toothed soricids has three layers; two of them are divided into two zones in the red‐toothed species. However, the most external layer varies among taxa; it is well defined in Sorex but difficult to identify in the Early Pleistocene genera Beremendia or Dolinasorex. In the arvicoline rodent Terricola, only two layers can be defined, the outer of which is divided into two zones depending on the presence or absence of Fe. The Fe proportions in the larger soricids reach up to 45%, but in rodents only up to 10% (weight % with respect to Fe + Ca + P). The STEM study shows that in a fossil soricid the Fe phases form clusters of nanometric particles of very poor crystalline oxides or hydroxides surrounding the apatite crystals that form the enamel.     

Macroscopic Sectioning of Undecalcified Tissues

An inexpensive laboratory apparatus designed to section undecalcified teeth and bone has been constructed. It consists of an electric motor with a mandrel bearing a carborundum sectioning disk centered within a Plexiglas enclosure. A coolant flows from a reservoir positioned in the upper portion of the Plexiglas enclosure to prevent desiccation or burning of the tissues. Undecalcified tissue can be cut into a series of thin, 0.5-1 mm slices to facilitate studies of pulpal enzymes, tooth morphology and design of dental cavity preparations.     

Mineral content of calcified tissues in cystic fibrosis mice

Although abnormal hard tissue mineralization is a recognized complication of cystic fibrosis (CF), the pathogenesis leading from the defective cystic fibrosis transmembrane conductance regulator (CFTR) protein is poorly understood. We hypothesized that CFTR plays a direct role in the mineralization of bone and teeth and tested the hypothesis using CF mouse models [CFTR(−) mice]. In vivo measurements by dual-emission X-ray absorpitometry (DEXA) indicated that bone mineral density (BMD) was reduced in CF mice as compared to gender-matched littermates. However, no change was evident after correction of BMD for the covariant of body weight. The latter finding was confirmed in isolated femurs and nasal bones by standard dry-ashing and instrumental neutron activation analysis (INAA). INAA of the continuously growing hypsodont incisor teeth from CFTR(−) mice revealed reduced Ca and normal P in the enamel layer—a finding consistent with changes in the deciduous teeth of CF children. Interestingly, enamel fluoride was increased in the CFTR(−) incisors and may associate with abnormal enamel crystallite formation. The iron content of the incisor enamel was reduced, explaining the loss of yellow pigmentation in CFTR(−) incisors. In contrast to the incisors, the mineral content of the slow-growing brachydont molar teeth was not different between CFTR(−) and CFTR(+) mice. It was concluded that CFTR does not play a direct role in the mineralization of bones or brachydont teeth in mice. Functional CFTR is apparently required for normal mineralization of the hypsodont incisors. However, multiple changes in the mineral composition of the CF incisors suggest an indirect role for CFTR, perhaps by maintaining a normal salivary environment for continuous tooth eruption. Preliminary reports published in Pediatric Pulmonology, 14, 253A (1997) and 15, 253A (1998).     

GROWTH LAYER PATTERNS IN TEETH FROM THE FRANCISCANA, PONTOPORIA BLAINVILLEI: DEVELOPING A MODEL FOR PRECISION IN AGE ESTIMATION

A model for estimating age from teeth of the franciscana, Pontoporia blainvillei , was developed using teeth from 72 specimens taken incidentally in fishing nets in Brazil and Uruguay. The goal of the model was to allow for accurate and precise age estimates for a larger sample of specimens. Growthlayer characteristics were selected on the basis of similarities in growth layers among various species of cetaceans, including those whose growth layers have been calibrated for time, and on length groups in the franciscana, which calves seasonally. Growth-layer patterns, including relative size and appearance, in both dentine and cementum did not vary by sex but did vary by the orientation of the tooth when it was sectioned. The model developed proved useful in providing guidelines for consistency in age estimation for biological studies of the larger sample of animals. We recommend the development of this type of model for other species to facilitate standardizing age estimation.     

Long-term potentiation in the anterior cingulate cortex and chronic pain

Glutamate is the primary excitatory transmitter of sensory transmission and perception in the central nervous system. Painful or noxious stimuli from the periphery ‘teach’ humans and animals to avoid potentially dangerous objects or environments, whereas tissue injury itself causes unnecessary chronic pain that can even last for long periods of time. Conventional pain medicines often fail to control chronic pain. Recent neurobiological studies suggest that synaptic plasticity taking place in sensory pathways, from spinal dorsal horn to cortical areas, contributes to chronic pain. Injuries trigger long-term potentiation of synaptic transmission in the spinal cord dorsal horn and anterior cingulate cortex, and such persistent potentiation does not require continuous neuronal activity from the periphery. At the synaptic level, potentiation of excitatory transmission caused by injuries may be mediated by the enhancement of glutamate release from presynaptic terminals and potentiated postsynaptic responses of AMPA receptors. Preventing, ‘erasing’ or reducing such potentiation may serve as a new mechanism to inhibit chronic pain in patients in the future.     

Maxillary palps can mediate taste rejection of plant allelochemicals by caterpillars

All caterpillars possess a pair of maxillary palps that “drum” the surface of foods during feeding. These chemosensory organs contain over 65% of a caterpillar's taste receptor cells, but their functional significance remains largely unknown. We examined their role in rejection of plant allelochemicals, using the tobacco hornworm (Manduca sexta) as a model insect and an extract from a plant species (Grindeliaglutinosa) as a model stimulus. We selected this system because hornworms reject foods containing Grindelia extract, and because preliminary studies indicated that their maxillary palps respond to this extract. We hypothesized that Grindelia extract elicits rejection through stimulating: (1) olfactory receptor cells, (2) taste receptor cells, (3) oral mechanoreceptors, and/or (4) a postingestive response mechanism. Our results were consistent only with hypothesis 2: caterpillars approached Grindelia-treated diets without apparent hesitation, but rejected it within 6 s of initiating biting; Grindelia-treated solutions stimulated taste receptor cells in the maxillary palp, but not the other gustatory chemosensilla; and ablating the maxillary palps eliminated rejection of Grindelia-treated diets. Our results demonstrate that taste receptor cells in the maxillary palps mediate rejection of Grindelia extract, and provide the first direct evidence for a role of maxillary palps in rejection of plant allelochemicals. Accepted: 25 January 1998