The Center for Membrane Toxicity Studies (CMTS), an NIEHS Marine and Freshwater Biomedical Sciences Center, was established at the Mount Desert Island Biological Laboratory in 1985.  The purpose of this Center is to involve a group of internationally recognized investigators, who are primarily experts in mechanisms of epithelial transport, to study the biological effects of environmental pollutants on cell and membrane transport functions. The primary emphasis of this research effort has been to elucidate the mechanisms of toxicity of environmental pollutants at the cellular and molecular level, using novel aquatic models developed at this laboratory.

The focus of the research programs of the Center has broadened in the last several years from the more narrow objective of identifying the molecular targets for the effects of heavy metals (or metal compounds) on cell functions, to include the effects of a broader range of environmental toxicants (including marine toxins) and the mechanisms by which the organism takes up and eliminates a wide range of xenobiotics and environmental pollutants. However, the concept that a “membrane lesion” accounts for the cellular toxicity of many environmental toxins still remains as a paradigm.

Annual Report

Download a copy of the 21st Annual Report of Progress, 2006 (pdf, 4MB)

Research Cores

Signal Transduction and Ion Transport

Investigators in the Signal Transduction and Ion Transport Core are examining the basic mechanisms concerning the cell’s signaling response to changes in its external environment, particularly as related to environmental stress, heavy metal exposure, marine toxins and environmental estrogens. These signaling pathways often involve mechanisms of homeostasis of ion transport, pH and cell volume regulation. Investigators in the Core are interested in determining the fundamental mechanisms by which cells regulate their cell volume, maintain internal pH and secretory functions and how these processes are disturbed by environmental influences.

Xenobiotic Transport and Excretion

Investigators in the Xenobiotic Transport and Excretion Core are examining the processes that are used by various epithelial tissues such as the liver and kidney to take up and excrete drugs and xenobiotics and other toxic compounds that enter from the environment and to study the effects of toxicants on this process. Investigators in this Core also interact with investigators working in the signal Transduction and Ion Transport Core.

Pilot Projects - 2006

Barbara S. Beltz, Ph.D., Professor, Department of Biological Sciences, Wellesley College
Neurogenesis and apoptosis in the crustacean brain: You are what you eat!

Shi-Ying Cai, Ph.D., Associate Research Scientist, Yale University School of Medicine
Bile salts modulate libid metabolism in Leucoraja erinacea, the little skate

H. Rex Gaskins, Ph.D., Professor of Immunobiology, Departments of Animal Sciences and Veterinary Pathobiology, Institute for Genomic Biology, University of Illinois at Urbana-Champaign
Development of Ciona intestinalis as a model for comparative toxicogenomics

Griselda Genovese, Ph.D., Postdoctoral Fellow, Department of Biodiversidad y Biologia Experimental, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires
Vitellogenin and Vitelline envelope proteins induction in Cichlasoma dimerus (Teleostei, Perciformes) exposed to waterborne octylphenol

Shawn E. Holt, Ph.D., Associate Professor, Department of Pathology, Massey Cancer Center, Medical College of Virginia at Virginia Commonwealth University
Understanding the regulation of telomerase and telomere biogenesis in marine animals

Alan C. Rigby, Ph.D., Assistant Professor of Medicine, Beth Israel Deaconess Medical Center
Marine snail conotoxins: Targeted probes for ion channels

David C. Sandeman, Ph.D., Professor, Department of Biological Sciences, Wellesley College
Adult neurogenesis in the crustacean brain: the effect of environment, locomotion, and toxins on the proliferation of neurons

Mary Voytek, Ph.D., Project Chief, United States Geological Survey
Environmental controls on bioavailability of mercury in estuarine systems: microbially mediated methylation and demethylation

Research Cores | Administration | Community Outreach and Education | Facilities

Administration

James L. Boyer, M.D., Center Director
Professor of Medicine, Yale University School of Medicine

Ned Ballatori, Ph.D., Associate Director
Professor of Environmental Medicine, University of Rochester School of Medicine

Patricia H. Hand, Ph.D.
Administrative Director, MDI Biological Laboratory

John N. Forrest, Jr., M.D.
Director, MDI Biological Laboratory
Professor of Medicine, Yale University School of Medicine

Claudine Lurvey
Director of Finance, MDI Biological Laboratory

Jerilyn M. Bowers
Director of Development and Public Affairs, MDI Biological Laboratory

Research Cores | Administration | Community Outreach and Education | Facilities

Community Outreach and Educational Programs (COEP)

Program Goals

The Center for Membrane Toxicity Studies (CMTS) promotes large and active community outreach and educational programs (COEP) based on the research and scientific findings of the work accomplished in conjunction with the Center.

• to broadly translate Center research findings into knowledge which can be applied to public health;
• to reach as many people, first locally, then nationally, as possible;
• to promote the use of unique marine models in environmental health science research
• to educate young people in environmental health science and inspire them towards a career in the discipline.

The major components of the CMTS COEP are

• Education, including Center-supported summer research programs for high school and undergraduate students interested in the environmental health sciences.
• Community Outreach, encompassing a summer visitors’ program supported by the Center, and hosting school groups to engage students in the work of the Center and environmental health in general.
• Community Environmental Health Laboratory (CEHL), a collaborative project of the Center and the Mount Desert Island Water Quality Coalition (MDIWQC), a local grassroots community organization conducting environmental monitoring in freshwater and marine environments on Mount Desert Island.
• Information Services an Annual Center Report, the dissemination of articles on Center activities and research breakthroughs to the local newspapers, radio and television, and the maintenance of a CMTS website.

Current Members

• Michael McKernan, COEP Director, MDIBL Director of Education and Conferences
• Jerilyn Bowers, COEP Associate Director, MDIBL Director of Development of Public Affairs

2006 Highlights

• 2006 student interns:  Center investigators mentored 20 student interns (6 high school, 14 undergraduate) and four teachers in the summer of 2006 in eight- to ten-week laboratory research projects and one-week research introductions for teacher participants. The students and teachers participated in hands-on research, including informal and formal laboratory meetings, and a weekly discussion group for high school students.  Through their mentor, students are trained and have full access to CMTS Facility Cores including Animal Services, Instrumentation, Cell Isolation, Culture and Organ Perfusion, Imaging, and Bioinformatics.  Students also attended CTMS enrichment activities including weekly seminars, conferences and symposia. 

At the end of the fellowship, all students write a research paper and complete a detailed program evaluation. In formative evaluations completed at the end of the fellowship year, students report high success and satisfaction with their laboratory experience.  For instance, 100% of the high school students surveyed (n=5) after the 2006 program said their personal objective were ‘entirely’ met - the highest mark in the category.  Four of five students said their ability to conduct research imporoved ‘extensively’ (the highest mark). One said ‘substantially’. High school students described their mentors as ‘approachable’, ‘knowledgable’, and ‘clear when instructing’. One student said “I was able to work at beginning stages of the experiment, something I hadn’t done before, and help design techniques with which to carry out later experiments”.

• Student Summit on Biomedical Research:  In March 2006, the Center co-sponsored with the Laboratory a Student Summit on Biomedical Research for 50 students from the gifted and talented programs of four local elementary schools. The Summit was framed around a hypothetical ‘new’ research laboratory that is to be built on Mount Desert Island. Student groups were instructed to study areas in biomedical research, prior to the Summit, then present and debate their various recommendations during the Summit.  Student-selected areas for study and consideration were: Environmental Health, Stem Cells, Gene Therapy, Cloning, Infectious Disease, Human Genome Project, Cardiovascular/Lung Disease, and Children’s Health. Prior to the Summit, students completed a webquest (http://www.mdibl.org/edu/webquest/), to help guide their study.  A board of scientific overseers (including investigators from MDIBL and The Jackson Laboratory), facilitated student presentations and debate.  Students also heard research presentations from Dr. Jim Denegre (The Jackson Laboratory), and Dr. James Coffman (MDIBL).  Lunch and laboratory tours were also provided.  Teacher evaluations indicated high satisfaction with Realization of Goals (on a scale of 1 to 5, a 5), Suitability of Activities (4.5), and WebQuest (5).  Student evaluations indicated high satisfaction with Overall Program (4.45), WebQuest (4), and Student Presentations (4.35).

• Community Environmental Health Laboratory (CEHL):  CEHL is a collaborative effort of the Center and the MDI Water Quality Coalition (MDIWQC).  In Spring 2006, CEHL hosted three students, two high school and one college student from Acadia University.  The students helped to collect baseline water quality and clam flat data at Hadley Point, an area slated for environmental restoration in 2007.  They also assisted with outreach projects to local schools which included red tide monitoring in all island middle schools, storm drain stenciling in three third grade classes in Bar Harbor, and clam flat surveys with Biology classes from MDI High School.  In May, CEHL sponsored the fourth Youth Watershed Forum, “Healthy Water Healthy People” at which 42 students from seven schools discussed water quality projects in oral and poster presentations.  Students participated in hands-on activities including phytoplankton tows, shoreline transects, and bacterial monitoring.  In addition to students and teachers, seven guests attended the forum along with 19 adult and youth facilitators.  The CEHL summer program, supported by the Center, was held June to August 2006.  An eight-week research internship was awarded to a local high school student who worked with four teacher interns over four weeks.   The student and teacher interns participated with CEHL lab director, Dr. Jane Disney on research projects including toxic phytoplankton monitoring, swim beach monitoring, and studies on the effects of cruise ship visitation on the water quality in Bar Harbor. As part of the Maine Healthy Beaches Program, the student intern worked with teacher interns to write weekly reports to local municipalities about the status of their beach monitoring efforts.  Final reports were prepared for town managers in Bar Harbor and Mt. Desert, ME and were presented at town meetings.  In Fall 2006, three students participated with the MDI Water Quality Coalition in continued studies around visiting cruise ships and continued to monitor for toxic phytoplankton species.  In addition, the students initiated a visioning project aimed at generating discussion about the future of Frenchman Bay and the possibility of a Bay Management Plan.

Research Cores | Administration | Community Outreach and Education | Facilities

Facilities

Animal Services Core | Instrumentation and Facilities Core
Cell Isolation, Culture and Organ Perfusion Core | Imaging Core | Bioinformatics Core

A. Animal Services

Overview
The objective of the Animal Services Core facility is to procure, care for and maintain in good health adequate numbers of the unique marine and freshwater animals used by the investigators at the Mount Desert Island Biological Laboratory.  These animals include:  spiny dogfish shark, skate, flounder, killifish and zebrafish, among others. CMTS members benefit from this Core Facility in terms of ease of access and preference in allocation of services and resources.

Core Director and Members

• George W. Kidder, III, Ph.D., Core Director; Senior Research Scientist, MDI Biological Laboratory
• Martha Ratner, Animal Services Core Technician

Equipment and Facilities
The Animal Service Core provides Center Investigators with healthy, fresh, local, marine species and maintains the highest level of care for these research animals.  The Laboratory's animal holding facility is comprised of 21 different circular tanks that range in size from 53 gallons (3' dia.) to over 5,297 gallons (15' dia.).  All tanks are 'flow-through' and are supplied with ambient temperature seawater pumped directly from the nearby ocean.  Freshwater or variable salinity systems are also available.  A full time technician provides animal care and monitors environmental conditions with the system.

Improvements during the past few years include:
 - the addition of new state of the art self-priming variable-speed seawater pumps with a capacity of 350 gallons of seawater/minute, more than twice that of the previous system; 
 - redesign and duplication of distribution piping throughout, which enables more efficient seawater delivery and allows  an entire one of the  circuits to be shut down at any time for cleaning; 
 - the construction of an elevated storage tank building with two 5000 gallon tanks for seawater to feed the distribution system.

Construction of a new marine aquarium facility featuring a 5 ft high, 16 ft diameter seawater fish tank for skates and sharks was completed in 2001.  Transparent panels provide viewing of the freely swimming animals.  The facility provides superior holding for the larger marine species as well as a venue for public education on the use of these unique marine models in toxicological research. 

Plans are currently underway for winterization of one of the fish holding buildings with the intention of making sharks and skates available at MDIBL year round.

Services Provided
Animal Procurement, Husbandry And Veterinary Care:
Procurement:  
 - The core contracts with a local fisherman. Sharks and skates are delivered to  15 ft. diameter, 5 ft. deep aquaria supplied with continuously flowing seawater.  Early in the season (May and June), before the shark migration reaches Maine, sharks are purchased from the Marine Biology Laboratory at Woods Hole, Massachusetts and trucked to MDIBL in large seawater totes.
 - Flounder are caught by hook and line from the shores of the Gulf of Maine by the animal intern and kept in large 6 ft. diameter holding tanks in continuously flowing seawater.  Killifish are caught in special seining traps in estuaries near the Laboratory on the change of the tides.  Research echinoderms and mollusks are collected fresh directly from the Laboratory shore and inter-tidal areas by the animal program intern and researchers or their technicians.
 - A zebrafish maintenance facility was put in place in 1997 with stock from a supplier in Florida.  The initial purpose of this colony is to provide zebrafish to investigators to familiarize themselves with the size and function of this increasingly interesting and useful species with the intention of inspiring innovative projects which might address toxicological issues from a genetic or developmental point of view.  Supply houses for genetically defined zebrafish will be a source of these animals as specific projects develop.

Husbandry  
 - Marine animals are maintained in aquariums with constantly flowing 15 degree C seawater.  In 2001 two 15 ft. diameter, one 10 ft. diameter and two 8 ft. diameter holding tanks were added to the animal holding facility.  An animal husbandry technician checks each holding area daily to assure proper seawater flow, fish density and food.  This person is responsible for quality control in keeping the aquariums clean and free of sick or dead fish.  He/she is the communication interface between the fisherman and the scientist, compiling orders for fish from the scientists, conveying these to the fisherman or other supplier and informing the scientists when fish have been delivered.

Veterinary care: One veterinarian services the animals within the CMTS core and assures that the animals are being maintained and used in an appropriate and humane manner.  The veterinarian advises CMTS research personnel to ensure that humane needs are met for the animals and are compatible with scientific requirements.  He oversees the adequacy of animal husbandry and nutrition, zoonosis control and hazards containment. The veterinarian also serves on the Institutional Animal Care and Use Committee which reviews all protocols  involving animal research in the toxicology center.

Compliance with all pertinent regulations:  The CMTS Animal Procurement and Holding Core is in compliance with the Animal Welfare Act of 1986 as spelled out in OPRR, Public Health Service Policy on Humane Care and Use of Laboratory Animals, 9/86, reprinted 3/96 and the MDIBL Animal Welfare Assurance #A3562-01 (expiration date June 30, 2004).  The Institutional Animal Care and Use Committee (IACUC) with its veterinarian member is the primary group responsible for carrying out the mandates of the Animal Welfare Act and the MDIBL Assurance.  Every animal protocol used  by CMTS investigators  must obtain IACUC approval before experiments begin.

Usage and Benefits
Center Investigators and students benefit from training offered by the Animal Services Core staff as follows:  
 - The Animal Care training program for humane practices, including training videos, demonstrations, animal welfare rules and regulations seminar, and a written test for animal handling certification are under the auspices of the CMTS animal procurement and holding core. 
 - Records animal use and experimental protocols; facilitates sharing of animal tissues; integrating ordering, receiving and billing of animal charges.
 - Proper disposal of animals and animal parts:  Utilization of MDIBL’s unique cold water marine animal models is critically dependent on the Animal Services Core.  

Examples of the marine animals utilized for research at MDIBL and this MFBS Center are listed below:

1) Spiny dogfish  (Squalus acanthias)
a) rectal glands used to study how salt secretion is disturbed in diseases such as cystic fibrosis  
b) rectal glands used to study the action of diuretics
c) rectal glands used to understand how cadmium toxicity occurs
d) rectal glands used to study the  transport of xenobiotics
e) heart muscle used to understand  how the heart regulates its rate and rhythm and how toxins affect it
f) aorta used to understand effects of heavy metals on regulation of hypertension 
g) testes used to study sperm cell development
h) choroid plexus is used to study xenobiotic transport in the brain
i) myocytes used to study the effects of maitotoxin on cardiac function
j) rectal glands used to study the effects of heavy metals on chloride channel function

2)  Little skate (Raja erinacea)
a) used to study how environmental pollutants accumulate and are metabolized
b) liver cells used to study how a marine toxin, microcystin, causes liver failure
c) liver and red blood cells used to study how cells regulate their own volume and how environmental pollutants disturb this function
d) egg capsules used to develop material for cartilage replacement 
e) liver and kidneys used to understand how environmental pollutants are removed from the body
f) multiple tissues used to study the molecular structure and function of ABC transporters, and other xenobiotic transporters
g) hepatocytes used to study the effects of toxicants on cytoskeleton proteins
h) hepatocytes used to express the effects of heavy metals on purigenic receptors and calcium signaling

3)  Winter Flounder (Pseudopleuronectes americanus)
a) used to study the actions of diuretics 
b) used to study gene expression when exposed to environmental toxicants
c) used to study the effects of heat shock and other stresses on renal organic and organic anion transport

4)  Killifish (Fundulus heteroclitus)
a)  used to study the effects of toxins on renal transport systems
b)  used to study how the eye is affected in diseases like glaucoma
c)  used to study gene expression after exposure to arsenic and other toxicants
d) used to study the transport of xenobiotics across brain capillaries

5)  Zebrafish (Danio rerio)
a) used to study neuroendocrine development
b) used  to study effects of endocrine disrupters
c) used to study the effects of environmental toxicants on reproduction and development

6) Xenopus oocytes (Xenopus laevis)
a)  used to study effects of maitotoxin non calcium and non-selective cation channels
b)  used to study the effects of heavy metals on chloride channel (CFTR) regulation

7) Sea Urchins (S.droebrachiensis and S. pupuratus)
a)  used to study effects of toxicants cell division and innate immunity

8) Adult Green Crab (Carcinius maenas)
a) used to study the effects of neurotoxins on neurogenesis and evoked nerve function

9) Tunicates (Ciona intestinalis)
a) used to identify novel regulatory elements in glutathione-s-transferases

Animal Services Core | Instrumentation and Facilities Core | Cell Isolation, Culture and Organ Perfusion Core | Imaging Core | Bioinformatics Core

Overview
The Instrumentation and Facilities Core objective is to provide CMTS Investigators basic equipment and services that are necessary to conduct their research.  Especially important are those items of essential equipment (such as refrigerated centrifuges, scintillation counters, cell culture incubators, etc.) which by virtue of size or expense would be unavailable unless provided by the MDIBL.  Several highly specialized facilities are also included in this Core including DNA sequencing and electrophysiologic facilities. The Instrumentation and Facilities Core also provides maintenance of equipment and training for equipment use for the staff of Center Investigators when necessary.  

The Instrumentation and Facilities Core is divided into 3 components: 1) Common equipment facilities including the Molecular Biology Facility, 2) A Marine DNA Sequencing Center providing rapid DNA sequencing (less than 24 hrs) essential for molecular analysis  3) An Electrophysiology facility that provides for oocyte injections and basic two electrode voltage recordings.

Core Director and Members

• Rolf Kinne, M.D., Ph.D., Core Director; Director, Max-Planck Institute, Dortmund, Germany
• George W. Kidder, Ph.D., Core Assoc. Director; Staff Instrumentation Officer; Senior Research Scientist, MDI Biological Laboratory
• Robert L. Preston, Ph.D., Core Assoc. Director; Professor of Physiology, Illinois State University
• Raymond A. Frizzell, Ph.D., Director, Electrophysiology & Oocyte Injection Facility; Professor and Chair, Department of Cell Biology and Physiology, School of Medicine, University of Pittsburgh
• Christine Smith, DNA Sequencing Center Technician, MDI Biological Laboratory
• David W. Towle, Ph.D., Director, DNA Sequencing Center; Senior Research Scientist, MDI Biological Laboratory
• Kathryn Schnackenberg, Analytical Technician
• Kasey Vermeulen, Analytical Technician

Equipment and Facilities
Section 1.
Services provided:
a. General Laboratory Equipment

• Analytical balances (5);  Mettler and  Sartorius                                                           
• Freezers, -20?C and -70?C (6)
• pH meters (3);  Beckman and Orion                                                                                   
• Spectrophotometer (1); Beckman                                                                                               
• Low speed centrifuges (2); Beckman DU 40                                                                       
• Superspeed refrigerated centrifuges (3); Sorvall                                   
• Ultracentrifuges (2); Beckman
• Scinitillation counter (1), Packard
• Isotope Disposal facilities                                                                                               
• Microplate reader (1), Dynatech                                                                                   
• Flame photometer (1); Instrumentation Labs                                               
• Lyophilizer (1) with Speedvac                       
• Osmometers (2); Wescor
• RO water purifiers (4), Barnstead                                               
• Ultra-pure water deionizers (4); Barnstead                                   
• Tissue homogenizers; Polytron and Omni                                                                                   

b.  Molecular Biology Equipment

• Autoclave (2)
• Cleanbench (1)
• Spectrophotometer (1).Beckman DU 640
• Eppindorf biophotometer
• Gel electrophoresis chambers and power supplies (2)           
• Thermocyclers (2), Perkin Elmer                                   
• Incubators (2)                                                                                               
• Gyrator (1)                                                                       
• Blotting apparatus (1)                                                                       
• Vacuum oven (1)                                                                                   
• Microwave (1)                                                                                               

Section 2.
DNA Sequencing Center
Director: Dr. David Towle

Services provided:

a = Sequencing
b = Oligo synthesis Ordering
c = Quantitative PCR
d = RNA BioAnalyzer
e = Microarray
f = ESTs

Section 3.
Electrophysiology and Oocyte Injection Center
Director: Dr. Raymond A. Frizzell           

a. Pipet/electrode fabrication
b. Microinjection
c. Xenopus oocytes
d. Two-electrode voltage clamp
e. Patch clamp recording
f. Analysis of electrophysiological data
g. Transepithelial impedance measurement and analysis

Usage and Benefits
1.  The General Equipment Facility provides members of the Center with facilities not readily available in the investigators' individual laboratories.  These include large items of equipment not easily transported to MDIBL on a seasonal basis.  This basic general use equipment is essential for all Center investigators at MDIBL.

2.  The Core provides equipment maintenance and inspection, assures compliance with Federal and State Radiation/Safety Regulations including disposal of radioactive and chemical waste and monitors and assures safe chemical storage.  

3.  The Core provides Molecular Biology and Tissue Culture facilities that permit application of the latest techniques in molecular biology to problems in toxicology.  This includes Western, Northern and Southern blotting, analysis of DNA/RNA purity, plasmid production, PCR and analysis of DNA fragments, gene expression, gel imaging and cloning. 

4.  The Core, through the agency of the Marine DNA Sequencing Center, provides on-site access to automated DNA sequence analysis; access to cDNA, mRNA, primers, tissue specific cDNA libraries and expression vectors.  The Marine DNA Sequencing Center has processed several thousand samples since it began operation six years ago.  The sequencing service is available year-round to all investigators for a small service charge. Investigator use is prioritized solely on a first-come, first-serve basis, although preference is given to CMTS members.  These functions are also related to the Cell Isolation, Culture and Organ Perfusion Core and the Imaging Core. 

5.  The Instrumentation and Facilities Core facilitates exchange of methods and approaches in environmental genomics, assists in analysis of DNA sequence data, data search in databases, information genomics and proteomics.  Of special interest are genes, proteins and regulatory pathways that are the targets of environmental pollutants and toxins.  MDIBL has established a  Bioinformatics Center under the direction of Carolyn Mattingly, Ph.D.,  Allan P. Davis, Ph.D., and Michael Rosenstein with the key objective of developing a Comparative Toxicogenomics Database.  The Database is being developed in cooperation with other MFBS Centers and will be a national repository for marine genomic and toxicological data.  This will be a unique resource that will serve national needs in this area.

6.  The objective of the Electrophysiology and Oocyte Injection Facility is to make available to members of the CMTS specialized equipment and expertise required to perform electrophysiological measurements on cells and tissues and to utilize the Xenopus oocyte as a heterologous expression system for understanding the effects of toxic substances on ion channel function. The core contains specialized equipment for oocyte injection, electrode and micropipette fabrication, two electrode voltage clamp, and patch clamp recording in the single channel configuration. Core personnel provide expertise for collaborative execution of pilot projects and service functions. 

The electrophysiology core is a natural point of interaction between investigators in the CMTS.  It is a natural point of collaboration between investigators with common interests and different expertise. Many investigators wish to study the expression of their protein of interest. With a permanent core facility, investigators can easily do this without the need of transporting this specialized equipment from their home laboratory and spending the time to set it up.  The same is true of two-electrode voltage clamp and patch clamp. Setting up a two-electrode voltage clamp setup can require several days of valuable time that can be saved by having a permanent core facility. Furthermore, investigators who want to try pilot projects can do this without the need of investing in a large amount of time setting up the equipment and learning the technique: they can collaborate with one of the core directors in doing the pilot project. 

Over the past several years, the Electrophysiology and Oocyte Injection facility has been used extensively by:

• John N. Forrest, M.D. (expression of shark CFTR and its Hg sensitivity) 
• Raymond A. Frizzell, Ph.D. (shark CFTR expression) 
• J. Denry Sato, Ph.D. (to assess CFTR function in oocytes)
• Bruce A. Stanton (two-electrode voltage clamp recordings to study the effects of cloned killifish SGK on CFTR Cl currents and the effects of arsenic).

Animal Services Core | Instrumentation and Facilities Core
Cell Isolation, Culture and Organ Perfusion Core | Imaging Core | Bioinformatics Core

C. Cell Isolation, Culture and Organ Perfusion Core

Overview
The NIEHS Center at the MDIBL enables investigators to utilize non-mammalian marine vertebrate and invertebrate species as experimental models to investigate aspects of environmental toxicology of relevance to human health. The overall goals of this Core Facility are to make these models more easily accessible to Center investigators, to encourage scientific collaborations, and to promote the sharing of expertise and resources.

The functions of the Core are: 1) to maintain the facilities necessary for cell and tissue culture, and organ perfusion, 2) to provide assistance to Center investigators in establishing or modifying specific cell culture or organ perfusion protocols, and 3) to provide cells in short-term and long-term primary culture, isolated tubules, and select cell lines to Center investigators. These services are provided free of charge to Center investigators, frequently on a collaborative basis, whereas other MDIBL investigators pay a small charge for these services. Center members also receive preference in the allocation of reagents and services, and in the use of the cell culture facilities. When the utilization of Core services by any one investigator becomes extensive, this individual is encouraged to establish these techniques in his/her own laboratory, and is provided guidance in doing so.

Core Director and Members

• David W. Barnes, Ph.D., Research Scientist; Director, Marine Cell Lines and Stem Cell Program, MDI Biological Laboratory
• Ned Ballatori, Ph.D., Core Assoc. Director;  Professor of Toxicology, University of Rochester School of Medicine
• Sarah Decker, Technical Assistant
• John N. Forrest, Jr., M.D., Core Assoc. Director; Director, MDIBL; Professor of Medicine, Yale University School of Medicine

Equipment and Facilities
The Cell Culture Facility Core provides expertise, equipment, and services related to established cell lines or primary cell cultures as well as the development and characterization of novel cell lines. This includes gene transfection and expression, in vitro assays, media formulation, microinjection, and cell banking. Many established animal cell lines are stored and available. The facility houses several inverted phase microscopes, equipped with epifluorescence systems, as well as laminar flow hoods, cooling, atmosphere-controlled incubators, a Coulter Counter, centrifuges, electroporation and electro-fusion equipment, and liquid nitrogen cell storage tanks. One research microscope has been fitted with a digital cooled camera capable of time-lapse photography. A micropipette puller, beveller and polisher for custom production of microinjection needles are also available. Also associated with the Core is a BD Biosciences Flow cytometer (FACSCallibur, with sorting capability and FACSStation data processing system. This system features electronics and optics for four-color display; software-controlled sorting in an aerosol-free sorting environment; single cell, exclusion and recovery sort modes; 300 cell/second capture rate; includes one-year warranty. FACSStation data processing system; power Macintosh G3/400MHz; CellQuest, FACSComp, FACSConvert, MODFIT and DNA Software; NEC 21" multisync color display; Lexmark laser printer; instrument interfaces. Facsloader multiple sample loader; Worklist Manager software, four loader carousels (40 tube capacity); and keypad control.

Below is a list of the cells and tissues that are made available through this Core, and a description of the administrative structure, location, and capability of each of these services.

1. Cell and Tissue Culture:
a. Skate hepatocyte primary cultures (suspension and monolayer)
b. Shark rectal gland isolated tubules
c. Shark rectal gland primary monolayer cultures, short and long-term
d. Flounder and killifish renal proximal tubules
e. Killifish brain capillaries
f. Shark choroid plexus
g. Cell lines from shark, skate, zebrafish, pufferfish, killifish, eel, salmonids, carp, cell cultures from invertebrate models (sea urchins, sea squirts, crustacea, snail)
h. CHO, COS, BALBC/3T3, T84 cells and a variety of other rodent, human and other mammalian cell lines

2.  Organ Perfusion:
i. Shark rectal gland
j. Skate liver

Usage and Benefits
The Core facilitates research and collaboration within the Center by maintaining facilities necessary for cell and tissue culture, and organ perfusion, providing assistance to Center investigators in establishing or modifying specific cell culture or organ perfusion protocols, and providing cells in short-term and long-term primary culture, isolated tubules, and select cell lines to Center investigators.  This has contributed to collaborative efforts in multiple areas. Some examples include the following projects:

• Structure and function and regulation of ABC transporters (Boyer, Ballatori, Mattingly Barnes).
• Renal excretion of organic solutes (Miller, Fricker, Henson).
• Mechanisms of chloride secretion and effects of heavy metals on the cytoskeleton in the shark rectal gland (Forrest).
• The cytoskeleton as a target for environmental toxicants (Miller, Henson, Barnes).
• Mechanisms of organic solute excretion by the liver (Boyer, Ballatori, Miller, Henson).
• Hepatocyte, brain and ovarian cultures for studies involving chronic administration of xenobiotics and gene regulation (Ballatori, Barnes, Henson, Miller, Boyer, Callard).
• Sulfur metabolism and gene expression in marine invertebrates (Gaskins, Barnes)
• Blood-brain barrier transport of foreign chemicals (Villalobos, Renfro, Miller)

The Core also has facilitated collaborations with investigators at other institutions outside the Center:

Dr. Chris Amemiya, Virginia Mason Institute, Seattle
Dr. Paul Collodi, Dept. of Animal Science, Purdue University
Dr. Steve Kaizainis, Wistar Institute, Philadelphia
Dr. James Ballantyne, Department of Zoology, University of Quelph, Canada 

In the last year the Core has responded to requests for materials or assistance from:

• Dr. Michael F. Romero, PhD, Dept. Physiology & Biophysics, Case Western Reserve University School of Medicine, Cleveland, OH 
• Dr Robert Parker, Division of Nutritional Sciences, Cornell University, NY
• Dr. Tomas Linder, Avd. för Metabola Sjukdomar, Karolinska Institutet, Sweden
• Dr. Lucia Rocco, Dept of Life Sciences, Second University of Naples, Italy
• Dr. Rebecca J. Van Beneden, Biochemistry and Marine Sciences, University of Maine, Orono
• Dr Edward Schmidt, School of Veterinary Medicine, University of Montana, Boseman
• Dr. Mark E. Hahn, Senior Scientist, Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA
• Dr. Nancy Kleckner, Department of Biology, Program in Neuroscience, Bates College, Lewiston, Maine
• Dr. David Mitchel, Department of Carcinogenesis, University of Texas
• Dr. Jeffery Bennetzen, Department of Genetics, University of Georgia, Athens, Georgia
• Dr. Rodney Nairn, M.D. Anderson Cancer Center, Smithville, Texas
• Dr. Marjorie Oleksiksiak, Department of Environmental  and  Molecular Toxicology, North Carolina State University

The Core has enhanced understanding of human environmental health
A major portion of the program associated with the Core is devoted to establishing transfection and cell culture technology for comparative marine biomedical models such as the little skate and spiny dogfish shark. In concert with the rapidly expanding genomic information available for marine models, in vitro approaches are essential to evaluate functionality of proteins and regulatory regions of these organisms. The human genome sequencing project has demonstrated the critical role of comparative genomics for interpreting the biomedical relevance of human mutations, polymorphisms and gene family relationships.

Future Plans
The future of predictive pharmacology and diagnostic medicine will depend on understanding genetic variability associated with human response to environmental components affecting disease states. As examples, work with CFTR in the isolated, perfused shark rectal gland and in rectal gland cell cultures is an example of a directly applicable model for studies of environmental toxicants on membrane proteins. Work in the core with cell cultures from Xiphophorus, a species of fish that is a classical model for studies of UV-induced melanoma, is of direct application to identifying human gene involved in predispositions to malignancies.

Educational Initiatives
This Core Facility has contributed to formal and informal training of new investigators and students in the basic procedures of organ perfusion, cell isolation, and tissue culture techniques. The facility has been used in several formal graduate and undergraduate courses taught at MDIBL.

Animal Services Core | Instrumentation and Facilities Core
Cell Isolation, Culture and Organ Perfusion Core | Imaging Core | Bioinformatics Core

D. Imaging Core

Overview
The Imaging Core provides the Center Investigator with state of the art microscopic imaging instrumentation and technology to further their research programs.  This Core also helps to organize and run a one-week, intensive Quantitative Fluorescence Microscopy course that covers all aspects of the technology, from the principals of fluorescence imaging to multidimensional imaging in living cells. Lectures dealing with the theory, mechanics, and application of fluorescent imaging methods are intermingled with extended laboratory practicals in which students are encouraged to use their own specimens, to optimize the utility of the course.

Core Director

• John H. Henson, Ph.D., Core Co-Director; Charles A. Dana Professor and Chair, Department of Biology, Dickinson College

Equipment and Facilities

• High resolution phase contrast, differential interference contrast, Hoffmann modulation contrast and conventional epifluorescence microscopy employing FITC, TRITC/Texas Red and DAPI channels.  Instruments available include a Nikon Optiphot 2 upright microscope, a Nikon Diaphot inverted microscope, an Olympus IM inverted microscope, and a Zeiss IM-35 inverted microscope.  
• Digitally enhanced real time and time lapse video microscopy. Equipment available includes Dage MTI newvicon, vidicon and CCD cameras, a Hamamatsu Argus 10 real time digital image processer, video frame import into Macintosh and PC computers employing either NIH Image or Metamorph image processing software, and conventional and time lapse super-VHS VCRs. 
• Low light level video microscopy employing Hamamatsu and Dage SIT, I-SIT or cooled-CCD cameras. 
• Automatic 35 mm microphotography using a Nikon UFX system. 
• High resolution video printing using Mitsubishi and Sony video copy processors.
• Quantitative image analysis with Power Macintosh and Pentium-based PCs using NIH Image/ImageJ and Metamorph software coupled with high capacity file storage devices.
• Confocal fluorescence microscopy - Olympus Fluoview Confocal Microscope - Model #B211
• Microspectrofluorometry - Photon Technology International system based on an Olympus inverted microscope, dual beam-grating based and tunable xenon light source and sensitive photomultiplier detection. The system is software-driven through a PC which also contains sophisticated data acquisition and analysis modules. Used for the ratiometric analysis of intracellular ion (e.g., Ca, Mg and pH) concentrations and capable of measuring very rapid (msec) changes.

Usage and Benefits

The Imaging Core is a major part of the research activities in the CMTS and a catalyst for collaborative projects among CMTS investigators (see below).  The centerpiece of the Core is an Olympus Fluoview Confocal Microscope.  This is one of only three confocal microscopes in the state of Maine.  This point source, dual laser (Ar and Kr) instrument is capable of imaging double-labeled samples in fluorescence mode as well as simultaneous transmitted bright field, phase or DIC.  Image analysis is further supported by specialized software within the Olympus package, free-standing Mac and PC image analysis workstations and a fast network with a server for image storge.  A high resolution, color printer is connected to the Olympus system.  The establishment of the Imaging Core has played a significant role in strengthening existing research collaborations among Center investigators and in developing new collaborative initiatives.  The areas of investigation that have benefited most from these imaging-based collaborations include:  

• studies on the cellular and molecular biology of xenobiotic excretion in liver, kidney, blood-brain barrier and elasmobranch rectal gland, 
• studies of the role of the cytoskeleton in ion transport, cell volume changes and volume regulation, 
• studies of cell signalling by calcium and marine toxins, 
• studies on xenoestrogen disruption of neural development.

2006 CMTS Users include:
David W. Barnes, Ph.D. - characterization of fish cell cultures.

Gert Fricker, Ph.D. Investigates mechanisms of xenobiotic excretion in renal proximal tubule, liver and choroid plexus. Collaborations with Boyer, Ballatori.

John H. Henson, Ph.D. - critical for characterizing the morphology and protein localization patterns in the sea urchin cells.

J. Denry Sato, Ph.D. - used confocal microscope to localize wild-type and mutant CFTR in a human cell line.

Bruce A. Stanton, Ph.D. - used the confocal microscope to study the effect of arsenic on MRP2 substrate transport in killifish proximal tubules

Barbara Beltz, Ph.D. and David Sandemann, Ph.D. - Their research project focused on the influence of environmental factors on adult neurogenesis in invertebrate models.  Invertebrate models were used because BrdU-laveled zones of proliferating central neurons are contained in small areas of the brain that can be visualized, assayed and quantified in their entirety in whole mounts.  To exploit this, and to achieve accurate cell counts, they depended heavily on the optical sectioning capabilities of the confocal microscope.
 
Other users of the Imaging Core:
John N. Forrest, Jr., M.D., Yale University School of Med., J.B. Claiborne, Ph.D., Georgia Southern University, David H. Evans, Ph.D., University of Florida

Course Benefit:
The seventh offering of a Quantitative Fluorescent Microscopy Course, headed by Dr. Simon Watkins of the University of Pittsburgh School of Medicine, once again brought numerous research and industrial sector experts and a great deal of state-of-the-art instrumentation to the MDIBL for an intensive one week session in June 2005. This definitely augmented the capabilities of the Imaging Core and several CMTS Center Investigators were involved in either teaching or taking this highly successful course. The course will again be offered in June of 2006 - the web site which describes the course is http://www.cbi.pitt.edu/qfm/index.html.

Animal Services Core | Instrumentation and Facilities Core
Cell Isolation, Culture and Organ Perfusion Core | Imaging Core | Bioinformatics Core

E. Bioinformatics Core

Objectives
The central goal of the Bioinformatics Core is to provide technical support for comparative studies of genes and proteins of toxicological significance. 

The functions of the Core are: 

• Develop the Comparative Toxicogenomics Database (CTD).   The goal of this project is to promote understanding about the effects of environmental chemicals on human health.  We are developing a publicly available database, the Comparative Toxicogenomics Database (CTD) to:  1) integrate data core to comparative toxicogenomics including references, sequences, genes, organisms, and chemical; 2) curating interactions between chemicals and genes and proteins; and 3) facilitating cross-species comparative studies of toxicologically important genes and proteins.  (CTD is supported independently by NIEHS (R33 ES011267), NCRR (P20 RR-016463), and the Mount Desert Island Biological Laboratory.

• Provide bioinformatics support to CMTS investigators.  Sequence analysis tools, training and storage resources in the Bioinformatics Core are available to Center members to conduct computational studies in toxicology.  Resources are freely available for Center members for analysis of toxicologically important genes and proteins with the agreement that results from these analyses will be integrated with data in CTD.

Members

• Carolyn J. Mattingly, PhD; Core Director; Director of Bioinformatics, MDIBL
• Allan Peter Davis, PhD; Scientific Curator, MDIBL
• Roy McMorran; System Administrator, MDIBL
• Michael Rosenstein; Scientific Software Engineer, MDIBL

Facilities and Equipment
Core Equipment:

Sequence Analysis Software (Commercial analysis tools were purchased and are supported by funds independent of the CMTS):

Usage and Benefits
The Bioinformatics Core engages collaborations with Center investigators conducting research on toxicologically significant genes and proteins by providing CTD and sequence analysis support.  Hypotheses generated from computational sequence analyses will be tested experimentally using other CMTS Cores including the Animal Services Facility, Instrumentation and Facilities and Cell Isolation, Culture and Organ Perfusion Cores. Results from collaborative studies will be integrated with data in CTD.  The Bioinformatics Core is currently working with the following Center investigators: Dr. James Boyer, Dr. Alice Villalobos, Dr. Larry Renfro, Dr. Bruce Stanton and Dr. David Barnes.

Educational Initiatives
The Bioinformatics Core has been involved in training students in basic bioinformatics and toxicology by participating in several formal undergraduate courses taught at MDIBL.

Animal Services Core | Instrumentation and Facilities Core
Cell Isolation, Culture and Organ Perfusion Core | Imaging Core | Bioinformatics Core

Research Cores | Administration | Community Outreach and Education | Facilities

Top of page