Ravindra M. Shah

Morphological, cellular, and biochemical aspects of differentiation of normal and teratogen-treated palate in hamster and chick embryos.

Publisher Summary This chapter focuses on some of the issues related to normal palate embryogenesis and drug-induced cleft palate pathogenesis in mammals and birds. Because this approach is significant from an evolutionary viewpoint, it may allow the correlation of evolutionary, embryological, and pathological aspects of palate development. The chapter focuses on normal and abnormal morphogenesis and differentiation at cellular, subcellular, and biochemical levels. Palatal morphogenesis is similar in hamsters and other mammals. The secondary palate originates as two vertical projections (shelves) on each side of the midline from the roof of the oronasal cavity. Two major points emerge from the experimental studies on two different classes of animals. First, normal palate development results from a preprogrammed series of events in cell populations of diverse embryological origin—a series that involves proliferation, interaction, differentiation, movement, and matrix synthesis. Second, normal palate development can be manipulated by affecting any of the preprogrammed series of events. Because of differences in their biological behavior, comparative studies of palatogenesis in the hamster and chick offer a unique opportunity to explore and manipulate these preprogrammed events.

Effects of 5-fluorouracil on collagen synthesis in the developing palate of hamster.

A study was undertaken to examine the effect of 5-fluorouracil (5-Fu) on collagen synthesis in the developing secondary palate. Pregnant hamsters were given 81 mg/kg 5-FU intramuscularly or 1 ml saline on day 11 of gestation. Control and treated embryonic palates, dissected from hamsters between days 11 and 13 of gestation, were incubated in a growth medium supplemented with [14C]proline. The rate of collagen synthesis, total protein and collagen isotypes were determined. The data showed that in control hamster palate the collagen synthesis peaked between days 12:00 (12 day: 0 h) and 12:04 of gestation, which is the period of shelf reorientation. In 5-FU-exposed hamster palates, the rate of collagen synthesis was lower than controls until day 12:04 of gestation followed by a rise on day 12:12 of gestation. In 5-FU-treated embryos palatal shelf reorientation took place between days 12:16 and 13:00 of gestation. Electrophoresis showed that only type I collagen was synthesized during palate development in both the control and 5-FU-treated hamster embryos. It was suggested that collagen synthesis may play a critical role in shelf reorientation in hamster since (i) new collagen was synthesized in both the control and 5-FU-treated hamster embryos prior to and during the period of normal reorientation, and (ii) in 5-FU-treated hamster embryos, a recovery in collagen synthesis precedes reorientation. Inhibition of collagen synthesis during abnormal development is only a step in the cascade of events of 5-FU-induced effects on protein synthesis.

In vivo/in vitro studies on the effects of cyclophosphamide on growth and differentiation of hamster palate.

A study was undertaken to examine the effects of cyclophosphamide (CP) on growth and differentiation of palatal tissues. An in vivo/in vitro approach was designed to analyze (1) whether the damage caused by in vivo administration of CP in the developing palate can be altered in vitro, and (2) to determine the effects of CP on the synthesis of collagen and glycosaminoglycan (GAG), which are essential for proper palate development. In addition, effects of vitamin B1 and/or B6 on in vivo modulation of CP teratogenicity was evaluated. Pregnant hamsters were given 30 mg/kg CP or 1 ml saline on day 10 of gestation. Control and CP-treated embryonic palates were dissected on day 11 of gestation and incubated in vitro in the presence or absence of CP. In order to allow metabolic activation of CP in vitro, either a slice of hamster liver or microsomal S9 fraction of liver was added to the culture medium. To study collagen and GAG synthesis, palates were obtained between days 10 and 13 of gestation, and incubated in growth medium supplemented with [14C]proline or [3H]glucosamine, as appropriate. The rates of collagen and GAG synthesis were determined. The results showed that, in the controls, the presence of a liver slice or S9 fraction in the culture medium had no effects on in vitro closure of palate. In vivo CP exposed palates did not fuse in vitro. When drug was given in vitro, or both in vivo and in vitro, palatal closure did not occur. CP reduced synthesis of both collagen and GAG in the vertically developing palate. The drug-treated shelves reoriented only after the rates of collagen and GAG synthesis were restored to the levels comparable to the control counterparts. Co-administration of vitamin B1 and B6 did not interfere with the teratogenicity of CP. It was suggested that CP treatment affected DNA synthesis and injured growing cells, which in turn reduced the synthesis of GAG and collagen and affected the expansion of shelf volume to delay the reorientation of the palatal shelves. Furthermore, it appears that in vivo treatment with CP changes the programming of palatal tissues to prevent the fusion process in vivo, which could not be altered in vitro.

Effects of EGF and TGF?1 onc-myc gene expression and DNA synthesis in embryonic hamster palate mesenchymal cells

Previous work has shown that cell proliferation is a major contributor to the early palate morphogenesis in mammals. The present study was undertaken to examine the effect of EGF, TGFβ1 and their combination on proliferation (measured by DNA synthesis) and on the expression of a growth related proto‐oncogene, c‐myc, in embryonic hamster palate mesenchymal cells (HPMC).

In vivo and in vitro assessment of mitogen activated protein kinase involvement during quail secondary palate formation

Spatiotemporally regulated cell proliferation and differentiation are crucial for the successful completion of morphogenesis of the vertebrate secondary palate. An understanding of the mechanisms by which these cellular phenomena are regulated during palate development involves the identification of the various signal transduction pathways. In the present study, the presence and activation of mitogen‐activated protein (MAP) kinases were investigated during the development of quail secondary palate. The palatal shelves were dissected on days 5–9 of incubation, homogenized, and centrifuged, after which the samples were separated by anion exchange fast protein liquid chromatography. The fractions were analyzed for myelin basic protein (MBP) phosphorylation. In addition, primary cultures of quail palate mesenchymal cells (QPMCs) were treated with epidermal growth factor (EGF) and prepared for MBP phosphorylation assays. A temporally regulated pattern of phosphotransferase activity, characterized by a three‐fold increase in phosphotransferase activity toward MBP between days 5 and 8 of incubation, was observed during quail palate development. Western blotting, using MAP kinase antibodies, demonstrated the presence of a 42‐kDa isoform between days 5 and 9 of incubation, during which the level of protein remained constant. Antityrosine immunoblotting with 4G10 also detected a 42‐kDa protein. Phosphotransferase assays, using either a MAP kinase‐specific substrate peptide (S5) or a protein kinase C inhibitor (R3), further confirmed the presence of a MAP kinase in the developing palate of quail. Because diverse biological processes occur concurrently during in vivo palate morphogenesis, the involvement of MAP kinase was explored further in primary cell culture. The data showed that EGF stimulated proliferation and activated 42‐kDa MAP kinase in QPMCs. It is suggested that MAP kinase cascade may be involved in growth factor‐regulated cell proliferation during morphogenesis of quail secondary palate. Anat. Rec. 252:194–204, 1998.

The Relationship Between Presence and Absence of Third Molars Hypodontia of other Teeth

Third molar agenesis is the most common form of hypodontia, occurring in 5-25% of the population (Banks, Angles Orthodont 4:223, 1934; Grahnen, Odont Rev 7:Suppl. 3, 1956; Garn and Lewis, Angles Orthodont 32:14, 1962). It has been repeatedly suggested that when one or more third molars are missing there is an increase in frequency of hypodontia of other teeth (Garn, Lewis and Vicinus, J Dent Res 41:717, 1962; Keene, Angles Orthodont 35:289, 1965). In the present communication we are reporting a survey of 7,886 Canadian individuals, in whom there was no association between congenitally missing third molars and agenesis of other tooth types. Intraoral radiographs and dental and medical histories of a series of 7,886 patients, registered at the Faculty of Dentistry, The University of British Columbia, Vancouver, were examined for presence or congenital absence of third molars and other teeth. From this sample, patients 20 years and older were selected. Only 653 cases were useful in the present study, the remainder being discarded because of youth, supernumerary teeth, impaction, history of early extraction, incomplete history regarding the status of missing teeth, and systemic illness during early childhood. Data collected were analyzed using the Student-t test method, for frequency, anatomical location, sex distribution and tooth type.

Changes in Casein Kinase 2 Activity During Development of the Secondary Palate in the Hamster

Casein kinase 2 (CK 2) is a serine/threonine kinase that has been ubiquitously conserved in all eukaryotic cells. The exact functions of this enzyme have not yet been clarified; however, studies have repeatedly suggested that it may play crucial roles in the regulation of cell proliferation. During the formation of the secondary palate in the hamster, bursts of cell proliferation occur during the initial half of vertical shelf development, which decrease during the subsequent steps of palate morphogenesis, thus indicating that the cell cycle in the developing vertical palate may be tightly regulated.

DEVELOPMENTAL ALTERATIONS IN CASEIN KINASE 2 ACTIVITY DURING THE MORPHOGENESIS OF QUAIL SECONDARY PALATE

During the progression of avian secondary palate morphogenesis, the rate of cell proliferation declines, whereas the production and accumulation of extracellular matrices increases. To investigate the regulation of these events, we examined the quail secondary palate for the activity of casein kinase 2 (CK 2), a pleiotropic serine/threonine second messenger independent enzyme implicated in cell growth and differentiation.

Toward Developing a Code of Ethics

A profession is considered to be ethical by its very nature. Hence, the ability to structure a set of principles that gives one a way to discuss the issues is crucial. However, numerous variables-such as culture, tradition of law, and stage of technological development of a Nation-State-confound issues in which principles may occasionally clash. There may not always be a right and wrong. Hence, for an international organization, a clear explanation of rules will be critical in the development of a code of ethics.

Effects of vincristine on developing hamster embryos.

Using dosages and a route of administration which resembled those used clinically in humans, the effects of vincristine on developing hamster embryos were evaluated. The results showed that the drug exerted a highly toxic effect in a dose-dependent manner; however, it exhibited only a sporadic teratogenic (gross malformation) effect. The data on DNA synthesis indicated involvement of internal organs and structures, which needs to be verified. Overall, the teratogenicity of the drug was more pronounced during pre-organogenesis than during the organogenesis period. It was suggested that, in contrast to the data reported in the literature, the different biological response to vincristine in hamster may relate to the use of the dose-route combination, and is potentially relevant to developmental and transplacental carcinogenesis studies.