Mark Wallert

Bringing the Excitement and Motivation of Research to Students; Using Inquiry and Research-Based Learning in a Year-Long Biochemistry Laboratory PART I-GUIDED INQUIRY-PURIFICATION AND CHARACTERIZATION OF A FUSION PROTEIN: HISTIDINE TAG, MALATE DEHYDROGENASE, AND GREEN FLUORESCENT PROTEIN.

A successful laboratory experience provides the foundation for student success, creating active participation in the learning process. Here, we describe a new approach that emphasizes research, inquiry and problem solving in a year‐long biochemistry experience. The first semester centers on the purification, characterization, and analysis of a novel fusion protein within a guided research experience. Throughout the semester, students gradually acquire skills as they are allowed to work independently. A fusion protein, malate dehydrogenase‐green fluorescent protein with a histidine affinity tag (MGH), is used throughout the semester. The fusion protein allows for a high throughput analysis and is stable for duration of the semester. Students start with the purification and analysis of the plasmid DNA and end with an enzymatic analysis of MGH. As students take ownership of their experiments and choose two different chromatographic resins, they make many choices throughout the semester. Skills, motivation, confidence levels, and attitudes were assessed before and after the semester. Students achieved high levels of critical biochemical laboratory skills and critical thinking while increasing their confidence and motivation for working in a biochemical research setting.

Sodium hydrogen exchanger and phospholipase D are required for α1-adrenergic receptor stimulation of metalloproteinase-9 and cellular invasion in CCL39 fibroblasts

Matrix metalloproteinase 9 (MMP-9) plays a critical role in digesting the extracellular matrix and has a vital function in tumor metastasis and invasion; this protease activity is significantly increased in non-small cell lung cancers. The sodium hydrogen exchanger isoform 1 (NHE1) functions as a focal point for signal coordination and cytoskeletal reorganization. NHE1 is thought to play a central role in establishing signaling components at the leading edge of a migrating cell. Therefore, we studied the relationship between NHE1 and MMP-9 activity in Chinese hamster lung fibroblasts (CCL39) stimulated with phenylephrine (PE). We show that PE increases MMP-9 gelatinolytic activity in CCL39 cells. The inhibition of phospholipase D (PLD) signaling abrogated PE-induced MMP-9 activity. The role of PLD as an essential signaling intermediate was confirmed when the addition of permeable phosphatidic acid increased MMP-9 activity in the same cells. PE-induced invasion was increased 1.9-fold over controls and the PE response was lost when 1-butanol was used to block PLD signaling. Cells pre-treated with the NHE1 inhibitor, 5-(N-ethyl-N-isopropyl) amiloride (EIPA) prior to PE addition resulted in a notable decrease in MMP-9 activation and cell invasion as compared to untreated PE-stimulated cells. CCL39 NHE1 null cells demonstrated no increase in MMP-9 protease activity or cell invasion in response to PE treatment. Reconstitution of NHE1 expression recovered the PE-induced activation of protease activity and cell invasion. MMP-9 processing was altered in cells expressing a proton transport defective NHE1 but retained the ability to respond to PE. Conversely, cells expressing an ezrin, radixin, moesin (ERM)-binding deficient NHE1 had a lower MMP-9 activity and the protease did not respond to PE addition. Parallel studies on NCI-H358 non-small cell lung cancer (NSCL) cells showed that PE stimulated both MMP-9 activity and cell invasion in an NHE1 dependent manner. This work describes for the first time a PE-induced relationship between NHE1 and MMP-9 and a new potential mechanism by which NHE1 could promote tumor formation and metastasis.

α1-Adrenergic Receptor Stimulation of Cell Motility Requires Phospholipase D-Mediated Extracellular Signal-Regulated Kinase Activation

Phospholipase D is suspected to play a role in tumorigenesis, and the inhibition of phospholipase D has been associated with changes in several cellular events including invasion and migration. We report here that the specific α1‐adrenergic receptor agonist, phenylepherine, signals to a growth factor pathway in a manner that requires phospholipase D activity in CCL39 fibroblasts. Phenylepherine increased extracellular signal‐regulated kinase phosphorylation eightfold and promoted stress fiber formation threefold. Stress fiber formation was blocked when extracellular signal‐regulated kinase activation was inhibited. Stimulation of CCL39 fibroblasts by phenylepherine increased the rate of wound healing fourfold in a wounding assay, while treatment with the MEK inhibitor, PD98059 reduced the closure of phenylepherine‐induced wound healing to control levels. Addition of 1‐butanol but not 2‐butanol inhibited extracellular signal‐regulated kinase activation by phenylepherine, presumably by blocking the formation of phosphatidic acid. Exogenously added cell permeable phosphatidic acid increased extracellular signal‐regulated kinase activation in a time‐ and dose‐dependent manner as well as stimulated the formation of stress fibers. 1‐butanol also significantly inhibited the ability of phenylepherine to stimulate stress fiber formation and wound healing. Taken together, these results indicate a novel role for phospholipase D in the activation of the extracellular signal‐regulated kinase growth factor pathway to stimulate early cellular events induced by phenylepherine.

RhoA Kinase (Rock) and p90 Ribosomal S6 Kinase (p90Rsk) phosphorylation of the sodium hydrogen exchanger (NHE1) is required for lysophosphatidic acid-induced transport, cytoskeletal organization and migration☆

The sodium hydrogen exchanger isoform one (NHE1) plays a critical role coordinating asymmetric events at the leading edge of migrating cells and is regulated by a number of phosphorylation events influencing both the ion transport and cytoskeletal anchoring required for directed migration. Lysophosphatidic acid (LPA) activation of RhoA kinase (Rock) and the Ras-ERK growth factor pathway induces cytoskeletal reorganization, activates NHE1 and induces an increase in cell motility. We report that both Rock I and II stoichiometrically phosphorylate NHE1 at threonine 653 in vitro using mass spectrometry and reconstituted kinase assays. In fibroblasts expressing NHE1 alanine mutants for either Rock (T653A) or ribosomal S6 kinase (Rsk; S703A) we show that each site is partially responsible for the LPA-induced increase in transport activity while NHE1 phosphorylation by either Rock or Rsk at their respective site is sufficient for LPA stimulated stress fiber formation and migration. Furthermore, mutation of either T653 or S703 leads to a higher basal pH level and a significantly higher proliferation rate. Our results identify the direct phosphorylation of NHE1 by Rock and suggest that both RhoA and Ras pathways mediate NHE1-dependent ion transport and migration in fibroblasts.

Kinetic analysis of glucose-6-phosphatase: an investigative approach to carbohydrate metabolism and kinetics

The enzyme glucose‐6‐phosphatase (G‐6‐Pase) catalyzes the hydrolysis of glucose‐6‐phosphate (G‐6‐P) to glucose. This is one of the key steps in gluconeogenesis and is critically important in maintaining stable blood glucose levels in most mammals. G‐6‐Pase is primarily found in the endoplasmic reticulum (ER) of hepatocytes and can easily be studied using isolated microsomes prepared from liver ER. A three‐part undergraduate laboratory exercise uses rat liver microsomes to focus on the enzymatic analysis of G‐6‐Pase. The assessment of G‐6‐Pase activity is conducted using a stopped assay protocol combined with a colorimetric determination of inorganic phosphate (Pi) levels. The laboratory exercise was designed to carry out an independent inhibition investigation using orthovanadate, a competitive inhibitor of G‐6‐Pase with potential clinical importance. The format of the three‐part investigation provides a useful mechanism for demonstrating enzyme kinetics and competitive inhibition using an enzyme that is important for carbohydrate metabolism and glycogen storage disease.

Implementing the recommended curriculum in biochemistry and molecular biology at a regional comprehensive university through a biology/chemistry double major: The minnesota state university moorhead experience*.

Minnesota State University Moorhead (MSUM) is a regional comprehensive university that is part of the Minnesota State Colleges and Universities (MnSCU) system. The current student population consists of ∼7,600 full‐ and part‐time students who are enrolled in one of 135 majors that lead to baccalaureate degrees. MSUM is committed to excellence in science teaching and research for undergraduates. It is an institutional member of the Council on Undergraduate Research and has three faculty members participating in Project Kaleidoscope (PKAL) Faculty for the 21st Century. Fourteen years ago, MSUM renewed its effort to have faculty participate in active research. All science faculty members hired since that time have been required to establish research programs. The primary purpose for the faculty engaging in ongoing research projects is to involve undergraduates in a meaningful research experience, thus training these students to become scientists.

You can Never have too many kinases: The Sodium Hydrogen Exchanger Isoform 1 Regulation by Phosphorylation

Regulation of Na+-H+ exchanger isoform 1 (NHE1) activity is a dynamic, integrated system demonstrating the complexity of kinase signaling. Studies of NHE1 have described 22 confirmed and putative phosphorylation sites on the cytoplasmic domain regulated by 12 protein kinases. However the final number of sites and the impact of these sites remains unclear. By identifying the Rock phosphorylation site and its role in regulating NHE1 activity, we began to understand the functional interplay between the RhoA/Rock and Rsk/Erk pathways. This demonstrates the need to have a complete and comprehensive understanding of the relationship between each kinase involved in NHE1 modification for a true understanding of the exchanger’s role in the control of cellular biological activity. By observing how the Rock phosphorylation site impacts cellular proliferation, it was demonstrated that Rock activity is only partially responsible for NHE1’s impact on biological activity. Furthermore, data presented here shows that Rock activity is involved in both α1-adrenergic (Phenylephrine) and LPA signaling but not PDGF activation of NHE1 transport activity suggesting that growth factor signaling does not require Rock, yet the kinase is essential in the effects of GPCR pathways. Of particular interest and adding to the complexity of kinase signaling, Pyk2 activity has been found to have an inhibitory effect on NHE1. These data demonstrate an uncertainty in the exact role that different kinases play in the regulation of NHE1 function, yet succeeds in providing evidence of the intricacy of phosphorylation organization and a need for further elucidation of the interplay between phosphorylation sites. Overall, the pivotal role of NHE1 in disease establishment and progression creates a solid argument for continued efforts in understanding its regulation.