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Recognizing the increasingly integrative nature of the molecular life sciences, the American Society for Biochemistry and Molecular Biology (ASBMB) recommends that Biochemistry and Molecular Biology (BMB) programs develop curricula based on concepts, content, topics, and expected student outcomes, rather than courses. To that end, ASBMB conducted a series of regional workshops to build a BMB Concept Inventory containing validated assessment tools, based on foundational and discipline-specific knowledge and essential skills, for the community to use. A culminating activity, which integrates the educational experience, is often part of undergraduate molecular life science programs. These "capstone" experiences are commonly defined as an attempt to measure student ability to synthesize and integrate acquired knowledge. However, the format, implementation, and approach to outcome assessment of these experiences are quite varied across the nation. Here we report the results of a nation-wide survey on BMB capstone experiences and discuss this in the context of published reports about capstones and the findings of the workshops driving the development of the BMB Concept Inventory. Both the survey results and the published reports reveal that, although capstone practices do vary, certain formats for the experience are used more frequently and similarities in learning objectives were identified. The use of rubrics to measure student learning is also regularly reported, but details about these assessment instruments are sparse in the literature and were not a focus of our survey. Finally, we outline commonalities in the current practice of capstones and suggest the next steps needed to elucidate best practices.

Undergraduate biology students are required to learn, understand and apply a variety of cellular and molecular biology concepts and techniques in preparation for biomedical, graduate and professional programs or careers in science. To address this, a simple laboratory module was devised to teach the concepts of cell division, cellular communication and cancer through the application of animal cell culture techniques. Here the mouse mammary tumor (MMT) cell line is used to model for breast cancer. Students learn to grow and characterize these animal cells in culture and test the effects of traditional and non-traditional chemotherapy agents on cell proliferation. Specifically, students determine the optimal cell concentration for plating and growing cells, learn how to prepare and dilute drug solutions, identify the best dosage and treatment time course of the antiproliferative agents, and ascertain the rate of cell death in response to various treatments. The module employs both a standard cell counting technique using a hemocytometer and a novel cell counting method using microscopy software. The experimental procedure lends to open-ended inquiry as students can modify critical steps of the protocol, including testing homeopathic agents and over-the-counter drugs. In short, this lab module requires students to use the scientific process to apply their knowledge of the cell cycle, cellular signaling pathways, cancer and modes of treatment, all while developing an array of laboratory skills including cell culture and analysis of experimental data not routinely taught in the undergraduate classroom.

The rat aromatic L-amino acid decarboxylase (AADC) gene contains alternative promoters directing expression of neuronal and nonneuronal mRNAs that differ only in their 5' untranslated regions (UTRs). We have analyzed the expression of the neuronal promoter of the AADC gene in cells synthesizing catecholamines and serotonin, as well as in non-AADC-expressing cells. We demonstrate the use of the neuronal-specific UTR in individual dopamine-, norepinephrine-, and serotonin-containing neurons. Transfection analyses show that the rat AADC neuronal promoter, containing 2,400 bp upstream of the transcription start site and including the 68-bp untranslated exon 2, can activate transcription from a reporter gene in both catecholaminergic and serotonergic cell lines. These analyses identified several positive and negative cis-active elements within this region. Unexpectedly, we observed that this promoter, when removed from its native context within the AADC gene, can also direct expression of a reporter gene in cells that do not normally express AADC mRNA. These results suggest that tissue-specific expression of the neuronal promoter may not be controlled by cis-active elements within the first 2,400 bp of the promoter. Additional information may be required to restrict neuronal promoter expression to appropriate cell types. This regulatory information could reside elsewhere within the promoter, within introns, or may be provided by interactions between the two AADC promoters.

Recognizing the increasingly integrative nature of the molecular life sciences, the American Society for Biochemistry and Molecular Biology (ASBMB) recommends that Biochemistry and Molecular Biology (BMB) programs develop curricula based on concepts, content, topics, and expected student outcomes, rather than courses. To that end, ASBMB conducted a series of regional workshops to build a BMB Concept Inventory containing validated assessment tools, based on foundational and discipline-specific knowledge and essential skills, for the community to use. A culminating activity, which integrates the educational experience, is often part of undergraduate molecular life science programs. These "capstone" experiences are commonly defined as an attempt to measure student ability to synthesize and integrate acquired knowledge. However, the format, implementation, and approach to outcome assessment of these experiences are quite varied across the nation. Here we report the results of a nation-wide survey on BMB capstone experiences and discuss this in the context of published reports about capstones and the findings of the workshops driving the development of the BMB Concept Inventory. Both the survey results and the published reports reveal that, although capstone practices do vary, certain formats for the experience are used more frequently and similarities in learning objectives were identified. The use of rubrics to measure student learning is also regularly reported, but details about these assessment instruments are sparse in the literature and were not a focus of our survey. Finally, we outline commonalities in the current practice of capstones and suggest the next steps needed to elucidate best practices.

Recognizing the increasingly integrative nature of the molecular life sciences, the American Society for Biochemistry and Molecular Biology (ASBMB) recommends that Biochemistry and Molecular Biolog...

We have developed a lab activity to teach the concepts of cell division, cellular communication and cancer, through application of proper animal cell culture techniques, to sophomore level biology majors. The lab module exploits the mouse mammary tumor cell line, MMT, as a model for breast cancer. Breast cancer, a disease involving unregulated cell proliferation, represents the second leading cause of cancer death in women. The drugs tamoxifen, curcumin, and NDGA are used routinely to treat female breast cancer. In this experiment, these three antiproliferative drugs are tested on MMT cells. Concentration of the drugs and length of the treatments are varied allowing students to determine the impact of these treatments on the rate of cell division. Students determine the best cell concentration for plating and growing cells, the optimal treatment time course, how to prepare and dilute drug solutions, and the rate of cell death vs cell division using simple cell counting protocols. The procedure lends to op...

Dopamine beta-hydroxylase catalyzes the final step in noradrenaline synthesis and is expressed exclusively in noradrenergic and adrenergic cells. In order to identify elements within the dopamine beta-hydroxylase (DBH) gene which contribute to the regulation of tissue-specific expression, we have analyzed the expression of the rat DBH promoter by transient transfection in both DBH-expressing and non-expressing cell lines. We have found that 1 kilobase of the DBH promoter can direct expression of the luciferase reporter gene in the DBH-expressing PC12, CATH.a, and SK-N-SH cell lines, but not in the non-DBH-expressing C6 glioma or CA77 cell lines. This activity was localized to a region between -133 and -173 upstream of the transcription start site. This element, however, also directed expression in non-DBH-expressing cell lines, but was inhibited when sequences between -212 and -388 were included. This inhibitory region contains sequences homologous to a silencer element recently identified in the human DBH gene, and shares homology with other previously identified silencer elements. Gel retardation experiments demonstrate that the rat DBH inhibitory region and the silencer elements found in the rat sodium type II channel and SCG10 genes bind a similar factor. The region between -133 and -173, which contains a consensus cyclic AMP response element (CRE), was also found to be responsive to cAMP in both DBH-expressing and non-expressing cells. Inclusion of sequences between -173 and -190 diminished the cAMP induction in PC12 cells, and nearly abolished the induction in C6 and CA77 cells, suggesting the presence of an additional negative element which inhibits cAMP induction in non-DBH expressing cells. DNA binding assays using antibodies to CRE binding protein-related transcription factors identified ATF-1 binding to the rat DBH-CRE, and further suggest that inhibition of cAMP regulation may be due to inhibition of ATF-1 binding by an additional factor, which binds to the DBH promoter immediately upstream of the CRE. These results demonstrate the importance of both positive and negative regulatory elements in the regulation of tissue-specific expression of the rat DBH gene.

The rat aromatic l-amino acid decarboxylase (AADC) gene contains alternative promoters which direct expression of neuronal and nonneuronal mRNAs that differ only in their 5'-untranslated regions (UTRs). We have analyzed the expression of the nonneuronal promoter of the rat AADC gene in the kidney epithelial cell line LLC-PK1 and in cells which do not express the nonneuronal form of AADC by transient transfection. These studies revealed that the first 1.1 kilobases of the nonneuronal promoter, including the nonneuronal-specific 5'-UTR (Exon 1), contains sufficient information to direct tissue-specific expression. Serial deletions of this promoter localized the cis-active element to a region between -52 and -28 base pairs upstream of the nonneuronal transcription start site. An A/T-rich sequence, within this region which we have termed KL-1, was found to bind a kidney and liver-specific factor by DNase footprint analysis and was capable of directing tissue-specific expression from a heterologous promoter. Moreover, when the KL-1 sequence was mutated in the context of the entire promoter sequence, all transcriptional activity was abolished. DNA sequence comparison revealed that the KL-1 fragment is highly homologous to the binding site for hepatocyte nuclear factor-1 (HNF-1). Mobility shift studies utilizing an antibody to HNF-1 demonstrated binding of HNF-1 to the KL-1 fragment and cotransfection of HNF-1 cDNA into cells which do not express the nonneuronal form of AADC resulted in activation of transfected AADC nonneuronal promoter constructs. These results strongly suggest that the transcription factor which regulates the tissue-specific expression of the nonneuronal form of AADC mRNA is HNF-1.

Aromatic L-amino acid decarboxylase (AADC, EC 4.1.1.28) catalyzes the decarboxylation of L-dopa to dopamine in catecholamine cells and 5-hydroxytryptophan to serotonin in serotonin-producing neurons. This enzyme is also expressed in relatively large quantities in nonneuronal tissues such as liver and kidney, where its function is unknown. Neuronal and nonneuronal tissues express AADC mRNAs with distinct 5' untranslated regions. To understand how this is accomplished at the genomic level, we have isolated rat genomic DNA encoding AADC. The organization of the AADC gene suggests that there are two separate promoters specific for the transcription of neuronal and nonneuronal forms of the AADC message. A small exon containing 68 bases of the neuronal-specific 5' end is located approximately 9.5 kilobases upstream of the translation start site, which is contained in the third exon. Approximately 7 kilobases upstream from the neuron-specific promoter is another small exon containing 71 bases of the 5' end of the nonneuronal AADC message. These data suggest that transcription initiating at distinct promoters, followed by alternative splicing, is responsible for the expression of the neuronal and nonneuronal forms of the AADC message.

Injury of the spinal cord leads to an inflammatory tissue response, probably mediated in part by cytokines. Because a common therapy for acute spinal cord injury is the use of an antiinflammatory synthetic glucocorticoid (methylprednisolone), we sought to determine mechanisms contributing to inflammation shortly after acute injury. Cytokine mRNAs [interleukin (IL)-1alpha, IL-1beta, tumor necrosis factor (TNF)-alpha, and IL-6] were increased during the first 2 hr following weight-drop compression injury by RNase protection assay, prior to the reported appearance of circulating lymphocytes. This immediate pattern of cytokine mRNA induction could be replicated in cultured, explanted spinal cord slices but not in whole blood of injured animals, which is consistent with a tissue source of cytokine mRNAs. Western blotting detected IL-1beta-like immunoreactivity released into culture medium following explantation and pro-IL-1beta-like immunoreactivity in freshly dissected spinal cord tissue. Pharmacologically blocking IL-1 and TNF-alpha receptors significantly reduced expression of IL-1alpha, IL-1beta, and TNF-alpha mRNAs. Finally, mice lacking both IL-1 and TNF-alpha receptors exhibited diminished induction of TNF-alpha, IL-6, and IL-1ra mRNAs following injury. Therefore, we conclude that contusion injury induces an immediate release of cytokines, which then contributes to the induction of cytokine mRNAs.