Research Interests
My approach to biological research has always crossed traditional boundaries with a strong conviction in the "comparative approach", be it at the molecular, cellular, or organismal level. I believe our knowledge of biological processes is too strongly biased by our willingness to accept the rat, mouse, or tumor cell line as "model systems".

The central question which drives my research is: "In what way are the fundamental structures and functions of living systems adaptively modified to allow organisms to exploit the diversity of habitiats and to maintain the radically different modes of life we observe in nature?" Along these lines there are two major research programs currently in our laboratory. The first involves elucidating the mechanisms of sex determination especially in those species without sex chromosomes. The second involves biochemical and physiological adaptations which allow the utilization of unique marine food sources, such wax esters and chitin.

Sex Determination
Sexual differentation into male or female is thought to depend on a switch mechanism. The switch can be chromosomally triggered based on chromosome counting (genotype sex determination or GSD) or environmentally triggered based on temperature or other environmental conditions (environmental sex determination or ESD). The mechanism of sex determination in egg-laying amniotes may be fundamentally different from that of the placental mammals. Differentiation of the manunahan ovary proceeds normally in the absence of estrogen whereas in birds and reptiles estrogen is essential for ovarian development. Recent data have shown that it is possible to sex reverse female embryos of birds and reptiles simply by blocking estrogen synthesis in the undifferentiated gonad. Conversely application of estrogen to male reptile embryos results in development of an ovary. These data suggest that the enzyme necessary for estrogen synthesis (CYP19, aromatase) in the developing gonad plays a critical role in sex determination in these vertebrates. We have begun an examination of the role and regulation of the aromatase gene in sex determination in two species of reptiles with TSD (temperature-dependent sex determination), the diamond back terrapin, Malaclemys terrapin and the American alligator, Alligator mississippiensis. These species were selected because (1) the sex of the embryos can be manipulated at will simply by setting incubation conditions, (2) extensive experience with successful handling of the eggs and embryos of these reptiles is at hand and (3) the temperature regimes that give rise to males or females differ markedly between the two species. Three full length cDNAs for the terrapin aromatase and a partial cDNA for the alligator aromatase have been obtained. The shortest of the cDNA constructs from the terrapin is capable of producing in vitro (coupled transcription/translation) as well as in vivo (transfected COS cells) a functional aromatase enzyme. In situ hybridization studies, as well as a competitive reverse transcription-polymerase chain reaction (RT-PCR) procedure, are being employed to discern the ontogeny of aromatase expression in these two reptiles. Sex determination in birds and reptiles may depend upon the initiation of estrogen synthesis in the indifferent gonad which inhibits male differentiation and stimulates ovarian development. In the absence of this estrogenic signal a testis develops. Future studies will address whether it is simply the activation of the aromatase gene or a gene or genes acting upstream from baromatase that is the initial trigger of the sex determining cascade in reptiles.

Biochemical Adaptation
The biochemical adaptations we are currently investigating involve assimilation of unique food items, such as wax esters and chitin by marine organisms. Marine wax esters (long-chain alcohols esterified to long-chain fatty acids) are synthesized in quantity by many marine animals, especially zooplankton, and are a major dietary component for many fish and seabirds. Whereas wax catabolism in some species of fish is reasonably well understood, wax utilization in higher vertebrates, especially in birds, has received little attention. We have recently shown in one species alcid and five species of petrels (all seabirds) that wax esters are rapidly broken down, apparently without the aid of gut flora, and deposited in storage fat depots as triacylglycerides. This is the first direct evidence that any seabird has the inherent capacity to digest and assimilate wax esters efficiently. These studies have been extended to include the yellow rumped warbler, a common North American passerine. With this species we have been able to show high assimilation efficiencies for not only wax esters but also bayberry wax (In both cases, assimilation efficiencies as high as 90% have been observed). Even beeswax was utilized with an efficiency of almost 25% to 75%. These findings are even more remarkable when one considers the transist time for meal in a yellow rumped warbler is only 1-3 hours. We have evidence that part of this capacity can be attributed to a bile-dependent pancreatic esterase which exhibits nearly identical rates of wax ester, cholesterol ester, and triacylglycerol hydrolysis. To better understand the physiological and biochemical basis of this unique adaptation we are currently purifying and characterizing this lipolytic activity. An additional finding was that the gallbladder in these avian species was able to attain bile salt concentrations as high as 0.6 molar, nearly 5 times that found in man.

We have estimated chitin digestibilities for Sooty Albatrosses (Phoebetria fusca), White-chinned Petrels (Procellaria aequinoctialis), Rockhopper Penguins (Eudyptes chrysocome), Gentoo Penguins (Pygoscelis papua), King Penguins (Aptenodytes patagonicus) and Leach's Storm-Petrels (Oceanodroma leucorhoa) fed Antarctic krill (Euphausia superba) and purified chitin. These species retain a substantial proportion (46.5 S.D. of 13.1%, 39.1 4.9%, 52.8 37.6, 45.3 5.6%, 84.8 11.7% and 35 12.2%, respectively) of ingested chitin. In order to assess the energetic and nutritional benefits of chitinolytic activity in seabirds, we studied gastrointestinal absorption of the end products of chitinolysis in Leach's Storm-Petrels. The overall absorption efficiency of NAG and its deacetylated precursor, glucosamine (Gln), in this species was 44.0 3.0% and 11.0 1.9%, respectively. These absorption efficiencies were significantly less that for glucose which was absorbed with an efficiency of 90.6 2.5%. No absorption of NAG and Gln occurred in the proventriculus. We also obtained preliminary estimates of chitinolytic activity in the gastric mucosae of the above six species by incubating extracts of tissue samples with a chitin substrate and measuring the production of the end-product of chitin hydrolysis, N-acetyl-D-glucosamine (NAG). Chitinolytic activity ( up to 5 000 g NAG h-I 9-1 expressed per g tissue) was measured from proventricular tissue and within the activity range (1350 to 61650 g NAG h-1g-1 ) reported for eight other avian species. No chitinolytic activity was recorded from pancreatic tissue or juices. The chitinolytic enzyme complex responsible for chitin hydrolysis in the stomach of rainbow trout (Oncorhynchus mykiss) has been successfully purified and partially characterized. Both a gastric lysozyme and a unique 42 kd chitinase appear to be responsible for initial chitin breakdown in the trout stomach. The 42 kd protein exhibited a specific activity of approximately 1 g NAG/hr g protein at 25 C and produced only chitobiose upon hydrolysis of reacetylated chitin. The temperature optimum for activity at pH 4.0 was 25 'C. We have obtained the N-terminal sequence of this protein and found it to be distantly related to the Brugia malayi microfilarial chitinase (10 out 14 matches) and the Manduca sexta chitinase (8 out of 10 matches).