The hamster, like the human produces cortisol as its major glucocorticoid, rather than corticosterone, typical of most enzyme rodents. It is not known, however, if the hamster cytochrome P450C17 (P450C17), a key enzyme for cortisol formation, also exhibits 17,20-lyase activity and if it catalyzes the formation of dehydroepiandrosterone (DHEA) at the adrenal level. To study this, we isolated the cDNA of P450C17 from a hamster adrenal library. This cDNA was sequenced and was found to have an open reading frame for a protein of 511 amino acids, as compared to the human P450C17, which contains 508 amino acids. The hamster P450C17 cDNA, in the coding region, is 76% homologous with the human P450C17 cDNA. The cDNA was then cloned in the expression vector pSV-SPORT 1, which was transiently transfected into COS 1 cells. The transfected cells were used for temporal studies on the transformation of radiolabeled C21-delta5- and C21-delta4-precursors. When transfected cells were incubated with [14C]pregnenolone, rapid formation of [14C]DHEA occurred. The intermediate 17alpha-hydroxypregnenolone accumulated initially with subsequent metabolism to DHEA. Likewise, when incubated with C21-delta4-steroids, [14C]progesterone and [3H]17alpha-hydroxyprogesterone, the 17,20-lyase product androstenedione was produced efficiently. In these studies, with respect to the delta5 pathway, the expressed hamster P450C17 gave similar results to bovine P450C17 cDNA inserted in the same expression vector. However, in contrast to the bovine enzyme, which converted low amounts of progesterone to androstenedione, the expressed hamster P450C17 enzyme showed an active metabolism via the delta4 pathway. Northern blot analysis, using the complete alpha-32P labeled hamster P450C17 cDNA as the probe, demonstrated a strong presence of P450C17 mRNA in hamster adrenals, a weaker presence in testes and ovaries, and no detectable species in brain, mesentery, and kidney. Immunoblotting analysis using an anti-rat P450C17 antibody demonstrated the presence of P450C17 protein in hamster adrenals, testes, and ovaries. Hamster adrenal cell suspensions and microsomal preparations were used to demonstrate the biosynthesis of [14C]17alpha-hydroxypregnenolone and [14C]DHEA from [14C]pregnenolone; both metabolites were formed during incubations. However, the ratio of [14C]DHEA/[14C]17alpha-hydroxypregnenolone was much lower in adrenal cells than in transfected COS 1 cells, indicating the presence of putative factors in hamster adrenal cells, favoring the 17alpha-hydroxylase activity rather than that of the 17,20-lyase. In conclusion, these studies demonstrate that the hamster adrenal is both a DHEA and a cortisol producer, and, therefore, this animal could be a suitable small animal model for the study of the role of DHEA in relation to human biochemistry and physiology.

The application of 3D printing and microcontrollers allows users to rapidly engineer novel hardware solutions useful in a laboratory environment. 3D printing is transformative as it enables the rapid fabrication of adapters, housings, jigs, and small structural elements. Microcontrollers allow for the creation of simple, inexpensive machines that receive input from one or more sensors to trigger a mechanical or electrical output. Bringing these technologies together, we have developed custom solutions that improve capabilities and reduce costs, errors, and human intervention. In this article, we describe three devices: JetLid, TipWaster, and Remote Monitoring Device (REMIND). JetLid employs a microcontroller and presence sensor to trigger a high-speed fan that reliably de-lids microtiter plates on a high-throughput screening system. TipWaster uses a presence sensor to activate an active tip waste chute when tips are ejected from a pipetting head. REMIND is a wireless, networked lab monitoring device. In its current implementation, it monitors the liquid level of waste collection vessels or bulk liquid reagent containers. The modularity of this device makes adaptation to other sensors (temperature, humidity, light/darkness, movement, etc.) relatively simple. These three devices illustrate how 3D printing and microcontrollers have enabled the process of rapidly turning ideas into useful devices.

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Western blot analyses of various hamster tissues reveal high levels of expression of 3 -hydroxysteroid dehydrogenase (3 -HSD) in adrenal and liver, and moderate levels of expression in kidney. The expression in liver is sexually dimorphic; very high levels of protein are observed in adult male liver but very low levels are seen in the female liver. Three distinct cDNAs encoding isoforms of 3 -HSD were isolated from hamster cDNA libraries. The type 1 isoform is a highaffinity dehydrogenase/isomerase expressed in adrenal and male kidney. The type 2 isoform is also a high-affinity dehydrogenase/isomerase expressed in kidney and male liver. The type 3 enzyme is a 3-ketosteroid reductase expressed predominantly in kidney. Sequencing of the clones showed that all three are structurally very similar, although types 1 and 2 share the greatest degree of similarity. Immunohistochemical staining for 3 -HSD in the adrenal was found throughout the adrenal cortex. In the kidney staining was confined to tubules, and in the liver, heavy staining was found in hepatocytes. The cloning of cDNAs for 3 -HSD from the liver and kidney should help in elucidating the function of this enzyme in these tissues.

19-Nor-corticosteroids are substances which have high mineralocorticoid activity and have been implicated in the development of essential hypertension. 19-Nor-deoxycorticosterone (19-nor-DOC) has been found in the urine of certain hypertensive patients suffering. However, very little is known regarding the origin and metabolism of 19-nor-DOC. Expression of the hamster adrenal cytochrome P450C11 cDNA in COS-1 cells has shown that this cytochrome has strong 19-hydroxylase activity, this activity being equivalent to that of 11beta-hydroxylase. Since one potential precursor of 19-nor-DOC is 19-hydroxy-deoxycorticosterone (19-OH-DOC), we have incubated this substrate in the presence of the hamster P450C11 expressed in COS-1 cells. We have found that the hamster P450C11 can transform 19-OH-DOC to 19-nor-DOC in high yield. These studies target, for the first time, the potential role of cytochrome P450C11 in the formation of 19-nor-DOC, a mineralocorticoid of adrenal origin that is possibly involved in the development of some types of hypertension.