Differential TRPV1 and TRPV2 channel expression in dental pulp.

Hypersensitivity to thermal and mechanical stimuli can occur in painful pulpitis. To explore the neuro-anatomical basis of heat and mechanical sensitivity, we evaluated expression of TRPV1 (heat) and TRPV2 (heat/mechanical) channels in the cell bodies and terminal arborizations of neurons that innervate the dental pulp (DP) and periodontal tissues (PDL). We report that ~50% of trigeminal ganglion (TG) neurons retrogradely labeled from the DP express TRPV2, and this was significantly greater than the general expression of this channel in the TG (15%) and slightly more than what is expressed in the PDL by retrograde labeling (40%). The TRPV1 receptor, however, was less prevalent in neurons innervating the DP than their general expression in the TG (17% vs. 26%) and was more extensively expressed in neurons innervating the PDL (26%). Co-labeling studies showed that 70% of neurons that innervate the DP are myelinated. Approximately 1/3 of the retrogradely labeled neurons from the DP were calcitonin-gene-related-peptide-positive (peptide-expressing), but very few expressed the IB4 marker of non-peptidergic unmyelinated afferents. These findings suggest that the DP has a unique neurochemical innervation with regard to TRP receptor expression, which has significant implications for the mechanisms contributing to odontogenic pain and management strategies.

Identification of cheese mite species inoculated on Mimolette and Milbenkase cheese through cryogenic scanning electron microscopy.

Samples of Mimolette (France) and Milbenkase (Germany) cheeses traditionally ripened by mites were analyzed to determine the mite species present on each sample. Scientific literature was reviewed to understand which mite species most commonly infest cheese. Morphological features possessed by mites were then studied to understand what unique characteristics are required to ensure accurate identification. After identification and compilation of a detailed key of stored food mites (subclass Acari, order Astigmata) and their delineating features, the mites were viewed through a cryogenic scanning electron microscope. It was determined that Mimolette cheese is inoculated with Acarus siro L. The features studied to identify this mite species included idiosomal length and shape, setae length and arrangement, leg size, placement of anus and genitals, and solenidia shape. The Milbenkase cheese is inoculated with Tyrolichus casei Oudemans, which was evident after viewing the same features used to identify A. siro and the supracoxal seta shape. With this knowledge, further research can be conducted on the 2 cheese varieties to understand what chemical, physical, and microbial changes occur within the cheeses because of mites. It is important to identify the mite species present on each cheese variety to improve our understanding of their role in creating the distinctive characteristics that set these cheeses apart from others.

Identification of non-functional human VNO receptor genes provides evidence for vestigiality of the human VNO.

In mammals, the vomeronasal organ (VNO) contains chemosensory receptor cells that bind to pheromones and induce a variety of social and reproductive behaviors. It has been traditionally assumed that the human VNO (Jacobson's organ) is a vestigial structure, although recent studies have shown minor evidence for a structurally intact and possibly functional VNO. The presence and function of the human VNO remains controversial, however, as pheromones and VNO receptors have not been well characterized. In this study we screened a human Bacterial Artificial Chromosome (BAC) library with multiple primer sets designed from human cDNA sequences homologous to mouse VNO receptor genes. Utilizing these BAC sequences in addition to mouse VNO receptor sequences, we screened the High Throughput Genome Sequence (HTGS) database to find additional human putative VNO receptor genes. We report the identification of 56 BACs carrying 34 distinct putative VNO receptor gene sequences, all of which appear to be pseudogenes. Sequence analysis indicates substantial homology to mouse V1R and V2R VNO receptor families. Furthermore, chromosomal localization via FISH analysis and RH mapping reveal that the majority of the BACs are localized to telomeric and centromeric chromosomal localizations and may have arisen through duplication events. These data yield insight into the present state of pheromonal olfaction in humans and into the evolutionary history of human VNO receptors.

Genomic characterization of the human heterotrimeric G protein alpha, beta, and gamma subunit genes.

Heterotrimeric guanine nucleotide binding proteins (G proteins) transduce extracellular signals received by transmembrane receptors to effector proteins. Each subunit of the G protein complex is encoded by a member of one of three corresponding gene families. Currently, 16 different members of the alpha subunit family, 5 different members of the beta subunit family, and 11 different members of the gamma subunit family have been described in mammals. Here we have identified and characterized Bacterial Artificial Chromosomes (BACs) containing the human homologs of each of the alpha, beta, and gamma subunit genes as well as a G alpha11 pseudogene and a previously undiscovered G gamma5-like gene. The gene structure and chromosome location of each gene was determined, as were the orientations of paired genes. These results provide greater insight into the evolution and functional diversity of the mammalian G protein subunit genes.

Contig assembly of bacterial artificial chromosome clones through multiplexed fluorescence-labeled fingerprinting.

A rapid multiplexed fingerprinting method has been developed for bacterial artificial chromosome (BAC) contig assembly. Defined subsets of BAC DNA fragments that result from digestion by three paired restriction endonucleases are labeled with unique fluorescent F-ddATP for each subset. Lists of the labeled fragment size are generated by an ABI 377 DNA sequencer and the GeneScan analysis software and then processed by an assembly program, FPC (Fingerprinted Contigs), to produce contig maps. Data obtained from the multiplexed labeling permit detection of smaller overlaps than is observed when data from a single double-digest are analyzed. The method has been tested on 98 BACs from chromosome 22 regions where large-scale sequencing is under way and also through simulation, using randomly generated BAC clones derived from existing DNA sequence data. In each case, contig assembly results demonstrated the advantages of multiplexed fingerprinting.

Karyotype analysis by computer and its application to mutagenicity testing of environmental chemicals.

Large scale population monitoring by cytogenetics would require vast amounts of effort for meaningful results. Computer techniques promise to assume some of this burden and thus render large scale monitoring a more practical alternative than it is now. A system recently developed at the California Institute of Technology, Jet Propulsion Laboratory and the City of Hope Medical Center, contains many of the features required by a large scale population monitoring system. One part of the system is a semi-automated slide preparation assembly which can process up to 576 specimens per day with uniform treatment. The second part of the system consists of a computer-controlled microscope which performs automatic slide search, metaphase location, and karyotype analysis under the interactive supervision of an operator. While the system was developed primarily for clinical cytogenetics, some aspects of our operating experience suggest promising approaches for population cytogenetics.