Faculty Bibliography

Recent studies suggest that the hypodigms representing the two earliest Australopithecus (Au. anamensis and Au. afarensis) form an ancestor-descendant lineage. Understanding the details of this possible transition is important comparative evidence for assessing the likelihood of other examples of ancestor-descendant lineages within the hominin clade. To this end we have analyzed crown and cusp base areas of high resolution replicas of the mandibular molars of Au. anamensis (Allia Bay and Kanapoi sites) and those of Au. afarensis (Hadar, Laetoli, and Maka). We found no statistically significant differences in crown areas between these hypodigms although the mean of M(1) crowns was smaller in Au. anamensis, being the smallest of any Australopithecus species sampled to date. Intraspecies comparison of the areas of mesial cusps for each molar type using Wilcoxon signed rank test showed no differences for Au. anamensis. Significant differences were found between the protoconid and metaconid of Au. afarensis M(2)s and M(3)s. Furthermore, the area formed by the posterior cusps as a whole relative to the anterior cusps showed significant differences in Au. afarensis M(1)s and in Au. anamensis M(2)s but no differences were noted for M(3)s of either taxon. Developmental information derived from microstructural details in enamel shows that M(1) crown formation in Au. anamensis is similar to Pan and shorter than in H. sapiens. Taken together, these data suggests that the overall trend in the Au. anamensis-Au. afarensis transition may have involved a moderate increase in M(1) crown areas with relative expansion of distal cusps.

The factor(s) regulating the combination of traits that define the overall life history matrix of mammalian species, comprising attributes such as brain and body weight, age at sexual maturity, lifespan and others, remains a complete mystery. The principal objectives of the present research are (1) to provide evidence for a key variable effecting life history integration and (2) to provide a model for how one would go about investigating the metabolic mechanisms responsible for this rhythm. We suggest here that a biological rhythm with a period greater than the circadian rhythm is responsible for observed variation in primate life history. Evidence for this rhythm derives from studies of tooth enamel formation. Enamel contains an enigmatic periodicity in its microstructure called the striae of Retzius, which develops at species specific intervals in units of whole days. We refer to this enamel rhythm as the repeat interval (RI). For primates, we identify statistically significant relationships between RI and all common life history traits. Importantly, RI also correlates with basal and specific metabolic rates. With the exception of estrous cyclicity, all other relationships share a dependence upon body mass. This dependence on body mass informs us that some aspect of metabolism is responsible for periodic energy allocations at RI timescales, regulating cell proliferation rates and growth, thus controlling the pace, patterning, and co-variation of life history traits. Estrous cyclicity relates to the long period rhythm in a body mass-independent manner. The mass-dependency and -independency of life history relationships with RI periodicity align with hypothalamic-mediated neurosecretory anterior and posterior pituitary outputs. We term this period the Havers-Halberg Oscillation (HHO), in reference to Clopton Havers, a 17th Century hard tissue anatomist, and Franz Halberg, a long-time explorer of long-period rhythms. We propose a mathematical model that may help elucidate the underlying physiological mechanism responsible for the HHO.

We propose a new classification of severe early childhood caries (S-ECC): hypoplasia-associated severe early childhood caries (HAS-ECC). This form of caries affects mostly young children living at or below poverty, characterized by structurally damaged primary teeth that are particularly vulnerable to dental caries. These predisposing developmental dental defects are mainly permutations of enamel hypoplasia (EHP). Anthropologists and dental researchers consider EHP an indicator for infant and maternal stresses including malnutrition, a variety of illnesses, and adverse birthing conditions. Differentiation of HAS-ECC from other forms of early childhood caries is warranted because of its distinct etiology, clinical presentation, and eventual management. Defining HAS-ECC has important clinical implications: Therapies that control or prevent other types of caries are likely to be less effective with HAS-ECC because the structural integrity of the teeth is compromised prior to their emergence into the oral cavity. By the time these children present to the dentist, the treatment options often become limited to surgical management under general anesthesia. To prevent HAS-ECC, dentists must partner with other health providers to develop interventions that begin with pregnant mothers, with the aim of eliminating or ameliorating the covariates accompanying poverty, including better pre- and post-natal care and nutrition.

Fully mature enamel is about 98% mineral by weight. While mineral crystals appear very early during its formative phase, the newly secreted enamel is a soft gel-like matrix containing several enamel matrix proteins of which the most abundant is amelogenin (Amelx). Histological analysis of mineralized dental enamel reveals markings called cross-striations associated with daily increments of enamel formation, as evidenced by injections of labeling dyes at known time intervals. The daily incremental growth of enamel has led to the hypothesis that the circadian clock might be involved in the regulation of enamel development. To identify daily rhythms of clock genes and Amelx, we subjected murine ameloblast cells to serum synchronization to analyze the expression of the circadian transcription factors Per2 and Bmal1 by real-time PCR. Results indicate that these key genetic regulators of the circadian clock are expressed in synchronized murine ameloblast cell cultures and that their expression profile follows a circadian pattern with acrophase and bathyphase for both gene transcripts in antiphase. Immunohistological analysis confirms the protein expression of Bmal and Cry in enamel cells. Amelx expression in 2-day postnatal mouse molars dissected every 4 hours for a duration of 48 hours oscillated with an approximately 24-hour period, with a significant approximately 2-fold decrease in expression during the dark period compared to the light period. The expression of genes involved in bicarbonate production (Car2) and transport (Slc4a4), as well as in enamel matrix endocytosis (Lamp1), was greater during the dark period, indicating that ameloblasts express these proteins when Amelx expression is at the nadir. The human and mouse Amelx genes each contain a single nonconserved E-box element within 10 kb upstream of their respective transcription start sites. We also found that within 2 kb of the transcription start site of the human NFYA gene, which encodes a positive regulator of amelogenin, there is an E-box element that is conserved in rodents and other mammals. Moreover, we found that Nfya expression in serum-synchronized murine ameloblasts oscillated with a strong 24-hour rhythm. Taken together, our data support the hypothesis that the circadian clock temporally regulates enamel development.

Transcellular bicarbonate transport is suspected to be an important pathway used by ameloblasts to regulate extracellular pH and support crystal growth during enamel maturation. Proteins that play a role in amelogenesis include members of the ABC transporters (SLC gene family and CFTR). A number of carbonic anhydrases (CAs) have also been identified. The defined functions of these genes are likely interlinked during enamel mineralization. The purpose of this study is to quantify relative mRNA levels of individual SLC, Cftr, and CAs in enamel cells obtained from secretory and maturation stages on rat incisors. We also present novel data on the enamel phenotypes for two animal models, a mutant porcine (CFTR-DeltaF508) and the NBCe1-null mouse. Our data show that two SLCs (AE2 and NBCe1), Cftr, and Car2, Car3, Car6, and Car12 are all significantly up-regulated at the onset of the maturation stage of amelogenesis when compared to the secretory stage. The remaining SLCs and CA gene transcripts showed negligible expression or no significant change in expression from secretory to maturation stages. The enamel of CFTR-DeltaF508 adult pigs was hypomineralized and showed abnormal crystal growth. NBCe1-null mice enamel was structurally defective and had a marked decrease in mineral content relative to wild-type. These data demonstrate the importance of many non-matrix proteins to amelogenesis and that the expression levels of multiple genes regulating extracellular pH are modulated during enamel maturation in response to an increased need for pH buffering during hydroxyapatite crystal growth.

Aim: Low bone mineral density and fragility fracture risk are said to be increased in HIV patients, yet the mechanism of action are unclear, in part because of limitations imposed by the non-invasive diagnostic methods employed. We aim to determine if there are bone mineral density and remodeling differences in midshaft femurs posthumously extracted from sub-Saharan Africans of Bantu origin with and without HIV/AIDS. Methods: 12 midshaft femurs derived from sub-Saharan Africans of known life history with (n=8) and without (n=4) HIV/AIDS were embedded in polymethylmethacrylate and sectioned. Quantitative backscattered electron microscopy (qBSE) in the scanning electron microscope (SEM) was performed with a Zeiss EVO 50 SEM.Contrast, and brightness conditions were standardized against two novel halogenated resins with mean atomic numbers bracketing the known low and high mineralization densities of bone. From 256 gray level images, bone mineral density variability was partitioned into eight equal grey-level bins and measured in proportion to the detected bone area using Leica Quantimet 550 image analysis software. Results: Contrary to previous studies, there is a tendency for increased bone mineral density around the midshaft femur cross section of individuals with HIV/AIDS, but this does not reach statistical significance in the small sample presently. However, the subpopulation of low density actively infilling osteons is low in HIV/AIDS individuals. Gray values between 0-160 reflect newly mineralizing low density bone, the proportion of which is reduced on average by 10% in the HIV/AIDS group. Mineral densities in the gray level range of 160-256 indicate older and more mineralized bone in remaining areas, which average 10% higher in the HIV/AIDS group. Conclusions: Bone remodeling is suppressed at the midshaft femur in HIV/AIDS individuals. The formation of new matrix is inhibited and bone packets are allowed to age and increase in mineral density, a phenomenon that we suggest may be linked to!

Huang Z, Kim J, Lacruz RS, Bringas P Jr, Glogauer M, Bromage TG, Kaartinen VM, Snead ML. Epithelial-specific knockout of the Rac1 gene leads to enamel defects. Eur J Oral Sci 2011; 119 (Suppl. 1): 168-176. (c) 2011 Eur J Oral Sci The Ras-related C3 botulinum toxin substrate 1 (Rac1) gene encodes a 21-kDa GTP-binding protein belonging to the RAS superfamily. RAS members play important roles in controlling focal adhesion complex formation and cytoskeleton contraction, activities with consequences for cell growth, adhesion, migration, and differentiation. To examine the role(s) played by RAC1 protein in cell-matrix interactions and enamel matrix biomineralization, we used the Cre/loxP binary recombination system to characterize the expression of enamel matrix proteins and enamel formation in Rac1 knockout mice (Rac1(-/-) ). Mating between mice bearing the floxed Rac1 allele and mice bearing a cytokeratin 14-Cre transgene generated mice in which Rac1 was absent from epithelial organs. Enamel of the Rac1 conditional knockout mouse was characterized by light microscopy, backscattered electron imaging in the scanning electron microscope, microcomputed tomography, and histochemistry. Enamel matrix protein expression was analyzed by western blotting. Major findings showed that the Tomes' processes of Rac1(-/-) ameloblasts lose contact with the forming enamel matrix in unerupted teeth, the amounts of amelogenin and ameloblastin are reduced in Rac1(-/-) ameloblasts, and after eruption, the enamel from Rac1(-/-) mice displays severe structural defects with a complete loss of enamel. These results support an essential role for RAC1 in the dental epithelium involving cell-matrix interactions and matrix biomineralization