Sequence analysis and function of mosquito aeCCC2 and Drosophila Ncc83 orthologs.

Dipteran insects have genes that code for two different Na+-dependent cation-chloride cotransporter (CCC) paralogs. Aedes aegypti aeNKCC1 is an ortholog of Drosophila melanogaster Ncc69, a bumetanide-sensitive Na+-K+-2Cl- cotransporter (NKCC). Aedes aegypti aeCCC2 and aeCCC3 are orthologs of Drosophila Ncc83. Prior work suggests that the transport properties of aeCCC2 differ from canonical NKCCs. In particular, Xenopus oocytes expressing aeCCC2 have increased Na+-dependent membrane currents compared to controls, whereas NKCCs are electroneutral. Here, we further evaluated the function and localization of aeCCC2 and Ncc83. In oocytes expressing aeCCC2 or Ncc83, membrane potential (Vm) hyperpolarized upon Na+ removal; following hypotonic exposure the change in Vm was greater than it was in controls. In voltage-clamp experiments, membrane currents were concentration dependent on Na+ with an apparent affinity (Km) of approximately 4.6 mM. In Malpighian tubules of larval and adult mosquitoes, aeCCC2 was localized along the basolateral aspect of principal cells. Sequence comparisons among transporters from Drosophila, Aedes, Anopheles, and Culex revealed 33 residues within the transmembrane domains (TMDs) that are fully conserved within paralogs but that differ between orthologs of NKCC1 and orthologs of aeCCC2/Ncc83. These residues are distributed across all 12 TMDs. Our results provide a foundation for further exploration of the structural basis for functional differences between insect Na+-dependent CCCs.

Mini-Review - Teaching Writing in the Undergraduate Neuroscience Curriculum: Its Importance and Best Practices.

In neuroscience and other scientific disciplines, instructors increasingly appreciate the value of writing. Teaching students to write well helps them succeed in school, not only because they perform better on assessments but also because well-structured writing assignments improve learning. Moreover, the ability to write well is an essential professional skill, because good clear writing in conjunction with good clear thinking results in increased success in fellowship applications, grant proposals, and publications. However, teaching writing in neuroscience classrooms is challenging for several reasons. Students may not initially recognize the importance of writing, teachers may lack training in the pedagogy of writing instruction, and both teachers and students must commit substantial time and effort to writing if progress is to be made. Here, we detail effective strategies for teaching writing to undergraduates, including scaffolding of teaching assignments, both within a class and across a curriculum; use of different types of writing assignments; early integration of writing into courses; peer review and revision of assignments; mentoring by student tutors; and use of defined rubrics. We also discuss how these strategies can be utilized effectively in the context of multicultural classrooms and labs.

Differential expression of putative sodium-dependent cation-chloride cotransporters in Aedes aegypti.

The yellow fever mosquito, Aedes aegypti, has three genes that code for proteins with sequence similarity to vertebrate Na+-K+-Cl- cotransporters (NKCCs) of the solute-linked carrier 12 superfamily of cation-chloride cotransporters (CCCs). We hypothesized that these mosquito NKCC orthologues have diverged to perform distinct roles in salt secretion and absorption. In phylogenetic analyses, one protein (aeNKCC1) groups with a Drosophila melanogaster NKCC that mediates salt secretion whereas two others (aeCCC2 and aeCCC3) group with a Drosophila transporter that is not functionally characterized. The aeCCC2 and aeCCC3 genes probably result from a tandem gene duplication in the mosquito lineage; they have similar exon structures and are consecutive in genomic DNA. Predicted aeCCC2 and aeCCC3 proteins differ from aeNKCC1 and vertebrate NKCCs in residues from the third transmembrane domain known to influence ion and inhibitor binding. Quantitative PCR revealed that aeNKCC1 and aeCCC2 were approximately equally expressed in larvae and adults, whereas aeCCC3 was approximately 100-fold more abundant in larvae than in adults. In larval tissues, aeCCC2 was approximately 2-fold more abundant in Malpighian tubules compared to anal papillae. In contrast, aeCCC3 was nearly 100-fold more abundant in larval anal papillae compared to Malpighian tubules, suggesting a role in absorption. Western blots with polyclonal antibodies against isoform-specific peptides revealed stronger aeCCC2 immunoreactivity in adults versus larvae, whereas aeCCC3 immunoreactivity was stronger in larvae versus adults. The differential expression pattern of aeCCC2 and aeCCC3, and their sequence divergence in transmembrane domains, suggests that they may have different roles in transepithelial salt transport.

Calcium binding to Procambarus clarkii sarcoplasmic calcium binding protein splice variants.

Sarcoplasmic calcium binding protein (SCP) is a high-affinity calcium buffering protein expressed in muscle of crayfish and other invertebrates. In previous work, we identified three splice variants of Procambarus clarkii SCP (pcSCP1a, pcSCP1b, and pcSCP1c) that differ in a 37 amino acid region that lies mainly between the 2nd and 3ed EF-hand calcium binding domain. To evaluate the function of the proteins encoded by the pcSCP1 transcripts, we produced recombinant pcSCP1 and used tryptophan fluorescence to characterize calcium binding. Tryptophan fluorescence of pcSCP1a decreased in response to increased calcium, while tryptophan fluorescence of the pcSCP1b and pcSCP1c variants increased. We estimated calcium binding constants and Hill coefficients with two different equations: the standard Hill equation and a modified Hill equation that accounts for contributions from two different tryptophans. The approaches gave similar results. Steady-state calcium binding constants (Kd) ranged from 2.7±0.7×10(-8)M to 5.6±0.1×10(-7)M, consistent with previous work. Variants displayed significantly different apparent calcium affinities, which were decreased in the presence of magnesium. Calcium Kd was lowest for pcSCP1a and highest for pcSCP1c. Site-directed mutagenesis of pcSCP1c residues to the amino acids of pcSCP1b decreased the calcium Kd, identifying residues outside the EF-hand domains that contribute to calcium binding in crayfish SCP.

Effect of body size on expression of Manduca sexta midgut genes.

Isometric growth of larval insect midgut predicts that the ratio of midgut surface area to body mass decreases as larvae grow. Gut tissue and gut content masses were measured in first through fifth instar Manduca sexta larvae. Wet mass of gut tissue increased in relationship to body mass with a scaling exponent of 0.85 compared to an exponent of 1.33 for gut content mass, suggesting that surface area becomes increasingly limiting in larger larvae. To test the hypothesis that compensation for the decrease in relative surface area of the midgut occurs by increased expression of membrane proteins, we compared midgut mRNA expression in fourth and fifth instar. Surveyed genes encoded apical membrane proteins with diverse functions, including the potassium amino acid transporter KAAT1, ion channel CAATCH1, aminopeptidase msAPN3, V-type H-ATPase E subunit, and cation chloride cotransporter masBSC. KAAT1 was expressed 300- to 1500-fold higher in middle and posterior midgut compared to anterior midgut. Expression of msAPN3 was approximately 200-fold higher in posterior midgut than middle midgut. Expression of KAAT1 was 2.3- to 3.1-fold higher in fifth compared to fourth-instar larvae, and masBSC expression was 1.3- to 1.9-fold higher in fifth-instar larvae. Expression of msAPN3 and V-ATPase, but not KAAT1, decreased as body mass increased within the fifth instar. Although the increased expression of KAAT1 and masBSC in fifth-instar larvae supports the hypothesis of increased membrane protein expression in larger larvae, results from the other genes do not support this hypothesis.

Characterization of sarcoplasmic calcium binding protein (SCP) variants from freshwater crayfish Procambarus clarkii.

Sarcoplasmic calcium binding protein (SCP) is an invertebrate EF-hand calcium buffering protein that has been proposed to fulfill a similar function in muscle relaxation as vertebrate parvalbumin. We have identified three SCP variants in the freshwater crayfish Procambarus clarkii. The variants (pcSCP1a, pcSCP1b, and pcSCP1c) differ across a 37 amino acid region that lies mainly between the second and third EF-hand calcium binding domains. We evaluated tissue distribution and response of the variants to cold exposure, a stress known to affect expression of parvalbumin. Expression patterns of the variants were not different and therefore do not provide a functional rationale for the polymorphism of pcSCP1. Compared to hepatopancreas, expression of pcSCP1 variants was 100,000-fold greater in axial abdominal muscle and 10-fold greater in cardiac muscle. Expression was 10-100 greater in fast-twitch deep flexor and extensor muscles compared to slow-twitch superficial flexor and extensors. In axial muscle, no significant changes of pcSCP1, calmodulin (CaM), or sarcoplasmic/endoplasmic reticulum Ca-ATPase (SERCA) expression were measured after one week of 4°C exposure. In contrast, large decreases of pcSCP1 were measured in cardiac muscle, with no changes in CaM or SERCA. Knockdown of pcSCP1 by dsRNA led to reduced muscle activity and decreased expression of SERCA. In summary, the pattern of pcSCP1 tissue expression is similar to parvalbumin, supporting a role in muscle contraction. However, the response of pcSCP1 to cold exposure differs from parvalbumin, suggesting possible functional divergence between the two proteins.

Cloning and characterization of a calmodulin gene (CaM) in crayfish Procambarus clarkii and expression during molting.

Calmodulin (CaM) is a highly conserved calcium (Ca(2+)) binding protein that transduces Ca(2+) signals into downstream effects influencing a range of cellular processes, including Ca(2+) homeostasis. The present study explores CaM expression when Ca(2+) homeostasis is challenged during the mineralization cycle of the freshwater crayfish (Procambarus clarkii). In this paper we report the cloning of a CaM gene from axial abdominal crayfish muscle (referred to as pcCaM). The pcCaM mRNA is ubiquitously expressed but is far more abundant in excitable tissue (muscle, nerve) than in any epithelia (gill, antennal gland, digestive) suggesting that it plays a greater role in the biology of excitation than in epithelial ion transport. In muscle cells the pcCaM was colocalized on the plasma membrane with the Ca(2+) ATPase (PMCA) known to regulate intracellular Ca(2+) through basolateral efflux. While PMCA exhibits a greater upregulation in epithelia (than in non-epithelial tissues) during molting stages requiring transcellular Ca(2+) flux (pre- and postmolt compared with intermolt), expression of pcCaM exhibited a uniform increase in epithelial and non-epithelial tissues alike. The common increase in expression of CaM in all tissues during pre- and postmolt stages (compared with intermolt) suggests that the upregulation is systemically (hormonally) mediated. Colocalization of CaM with PMCA confirms physiological findings that their regulation is linked.

Elongation factor 1Bgamma (eEF1Bgamma) expression during the molting cycle and cold acclimation in the crayfish Procambarus clarkii.

Eukaryotic elongation factor 1Bgamma (eEF1Bgamma) is a subunit of elongation factor 1 (EF1), which regulates the recruitment of amino acyl-tRNAs to the ribosome during protein synthesis in eukaryotes. In addition to structural roles within eEF1, eEF1Bgamma has properties which suggest sensory or regulatory activities. We have cloned eEF1Bgamma from axial abdominal muscle of freshwater crayfish, Procambarus clarkii. The predicted amino acid sequence has 66% identity to Locusta migratoria eEF1Bgamma and 65% identity to Artemia salina eEF1Bgamma. We measured eEF1Bgamma expression by real-time PCR, using the relative quantification method with 18s ribosomal RNA as an internal calibrator. eEF1Bgamma expression was lowest in gill, axial abdominal muscle, and hepatopancreas, and was highest in the antennal gland (5.7-fold above hepatopancreas) and cardiac muscle (7.8-fold above hepatopancreas). In axial abdominal muscle, eEF1Bgamma expression was 4.4-fold higher in premolt and 11.9 higher in postmolt compared to intermolt. In contrast, eEF1Bgamma was decreased or unchanged in epithelial tissues during pre- and postmolt. eEF1Bgamma expression in the hepatopancreas was 3.5-fold higher during intermolt compared to premolt and was unchanged in gill and antennal gland. No significant differences in eEF1Bgamma were found after 1 week of acclimation to 4 degrees C. These results show that eEF1Bgamma is regulated at the mRNA level with tissue-specific differences in expression patterns.

Paradox of epithelial cell calcium homeostasis during vectorial transfer in crayfish kidney.

The molting cycle of the freshwater crayfish, Procambarus clarkii, has been used as a model to study the cellular physiology and molecular biology of Ca "supply" proteins that effect transcellular vectorial Ca(2+) movement to achieve organismal Ca homeostasis. Specifically, periods of net Ca(2+) influx (postmolt) have been compared with periods of net Ca(2+) balance (intermolt). The broader goal is to understand the paradox facing epithelial cells of maintaining low cytosolic Ca(2+)in the face of mass Ca(2+)transit across epithelial cells. This mini-review compares mRNA and protein expression profiles for a series of proteins that are of strategic importance in effecting transcellular Ca(2+) flux in a selected epithelium, the antennal gland (kidney analog) specifically during apical to basolateral Ca(2+) conveyance. Target proteins were selected as representative of key "stages" in the transcellular transfer of Ca(2+): import (epithelial Ca(2+) channel, ECaC); storage (sarco/endoplasmic reticulum Ca(2+) ATPase, SERCA); buffering (sarcoplasmic Ca(2+) binding protein, SCP); and export (plasma membrane Ca(2+) ATPase, PMCA and Na(+)/Ca(2+) exchanger, NCX). The purpose of this review is to assess coordination of expression of these target proteins at times of high Ca(2+) demand (premolt and postmolt) compared to low Ca demand (intermolt) as a function of cellular location (apical vs. basolateral; endomembranes vs. plasma membranes) and relative abundance within different regions of the antennal gland. Understanding the spatiotemporal regulation of Ca(2+) handling proteins involved in transcellular transport is fundamental to investigating their endocrine regulation.

Criticism and interpretation: teaching the persuasive aspects of research articles.

Research articles are an excellent tool for promoting active learning about the scientific process. One difficulty in teaching research articles is that they address a professional audience and often seek to be persuasive as well as informative. This essay discusses pedagogical strategies that are intended to help students differentiate the purely informative aspects of research articles, such as descriptions of the methods and results, from the persuasive aspects, such as interpretation of results and critical evaluation of the work of other scientists.

Molecular characterization of the sarcoplasmic calcium-binding protein (SCP) from crayfish Procambarus clarkii.

Sarcoplasmic Calcium-binding Protein (SCP) is believed to function as the invertebrate equivalent of vertebrate parvalbumin, namely to "buffer" cytosolic Ca2+. We have cloned and characterized a novel SCP from axial abdominal muscle of crayfish Procambarus clarkii (referred to as pcSCP1), and have examined tissue specific distribution and expression as a function of molting stage in non-epithelial and epithelial tissues. The complete sequence of pcSCP1 consists of 1,052 bp with a 579 bp open reading frame, coding for 193 amino acid residues (molecular mass of 21.8 kDa). There is a 387 bp 3' terminal non-coding region with a poly (A) tail. The deduced pcSCP1 protein sequence matched most closely with published SCP sequences from another crayfish Astacus leptodactylus (92.8%) and from shrimp (78.6-81.2%) and fruit fly (53%). Real-time PCR analysis confirmed that pcSCP1 is ubiquitously expressed in all tissues tested (gill, hepatopancreas, intestine, antennal gland, muscle); however it is most abundant in muscle particularly in the axial abdominal muscle. The real-time PCR analysis revealed that pcSCP1 expression is downregulated in pre- and postmolt stages compared with intermolt. Epithelial (hepatopancreas and antennal gland) SCP expression exhibited a more dramatic decrease than that observed in muscle. Expression trends for pcSCP1 paralleled published trends for sarco/endoplasmic reticular calcium ATPase (SERCA), suggesting that their cellular function in regulating intracellular Ca2+ is linked.

The cation-chloride cotransporter, masBSC, is widely expressed in Manduca sexta tissues.

Cation-chloride cotransporters, including the Na-K-Cl cotransporter, play an important role in epithelial ion transport in insects. We have determined the tissue distribution of Manduca sexta bumetanide sensitive cotransporter (masBSC), a putative Na-K-Cl cotransporter that was originally cloned from M. sexta Malpighian tubules. We developed a polyclonal antibody (M6) against a C-terminal fragment of masBSC. masBSC protein was detected by M6 at an apparent molecular mass of approximately 220kDa in M. sexta foregut, midgut, hindgut, Malpighian tubule, salivary gland, fat body, trachea, and nerve cord. Higher expression was observed in the foregut than in other tissues. M6 stained the apical membrane of midgut epithelial cells in cross-sections of third instar larvae. The transcript of masBSC was detected by RT-PCR in midgut, Malpighian tubule, hindgut, trachea, nerve cord, and fat bodies. Taken together, these findings demonstrate that masBSC is widely expressed in M. sexta. While the specific function of masBSC remains unknown, the wide distribution indicates a role of masBSC in a broad range of tissues.

An online tutorial for helping nonscience majors read primary research literature in biology.

Using primary literature is an effective tool for promoting active learning and critical thinking in science classes. However, it can be challenging to use primary literature in large classes and in classes for nonscience majors. We describe the development and implementation of an online tutorial for helping nonscience majors learn to read primary literature in biology. The tutorial includes content about the scientific process and the structure of scientific papers and provides opportunities for students to practice reading primary literature. We describe the use of the tutorial in Biology of Exercise, a course for nonscience majors. Students used the tutorial outside of class to learn the basic principles involved in reading scientific papers, enabling class sessions to focus on active-learning activities and substantive class discussions.

Reduced blood-to-tissue albumin movement after plasmapheresis.

We tested the hypothesis that a decrease in the blood-to-tissue movement of albumin contributes to the recovery of plasma albumin and plasma volume after acute plasma protein depletion (plasmapheresis). Awake and unrestrained male Sprague-Dawley rats (220-320 g) fitted with jugular catheters were plasmapheresed, and plasma volume, plasma albumin, and total plasma protein content were measured at 1, 5, 24, and 48 h postplasmapheresis. Plasma volume recovered to baseline within 1 h (4.6 +/- 0.42 vs. 4.7 +/- 0.46 mL/100 g body weight (bw), remained at baseline from 5 h to 24 h but increased to 5.5 + 0.57 mL/100 g bw at 48 h (P < 0.05). Plasma albumin and total protein content recovered rapidly but remained below baseline levels at 1 h (10.05 +/- 0.98 vs. 12.33 +/- 1.29 and 19.75 +/- 1.75 vs. 24.73 +/- 2.56 mg/100 g bw, respectively). Plasma protein content retumed to baseline by 5 h of recovery. Tissue uptake of I125-labeled albumin decreased in the heart, skin, skeletal muscle, and small Intestines of plasmapheresed rats (P < 0.05). These data support the hypothesis that a reduction in albumin efflux from the vascular space contrlbutes to the recovery of plasma albumin and total protein content during plasma volume recovery and eventual expansion after plasmapheresis.