Joseph R. Koke

Summer Scientific Research for Teachers: The Experience and its Effect

Doing science – conducting actual research side by side with researchers—is perhaps the best way to achieve scientific literacy (Duchovnay andJoyce, 2000)IntroductionScientific inquiry is fundamental to the science education reform movement.It is the dominant theme of the 1990s reform documents such as the NationalScience Education Standards [NSES] (National Research Council, [NRC], 1996a)and the Benchmarks for Science Literacy (Association for the Advancement ofScience [AAAS], 1993). The development of scientific inquiry skills is consideredto be the most effective way to create a society of scientifically literate citizens(NRC, 1996a). As stated in the NSES, “when engaging in inquiry, students...actively develop their understanding of science by combining scientific knowledgewith reasoning and thinking skills” (NRC, 1996a p. 2). Inquiry methods oftenaddress issues that are important to the community such as environmental andhuman health. It is believed that students will be better able to discuss issues andmake informed decisions about scientific issues if they have developed reasoningskills through inquiry methods.Teachers who have learned science facts from textbooks and lectures, butlack scientific research experiences, may have difficulty teaching science by inquiry(Hodson, 1993; Roth, McGinn, and Brown, 1997). Beisenherz & Dantonio (1991)stated that, “teachers cannot be lectured at, demonstrated to, and asked toThis material is based upon work supported in part by gifts and grants from the NationalScience Foundation (ESI #9731321), the Eisenhower Professional Development GrantsProgram (#98024) administered by the Texas Higher Education Coordinating Board,and the Southwest Bell Corporation Foundation. Any opinions, findings, andconclusions or recommendations expressed in this article are those of the author(s)and do not necessarily reflect the views of the funding agencies.

Activating transcription factor 3 (ATF3) expression in the neural retina and optic nerve of zebrafish during optic nerve regeneration

Fish, unlike mammals, can regenerate axons in the optic nerve following optic nerve injury. We hypothesized that using microarray analysis to compare gene expression in fish which had experienced optic nerve lesion to fish which had undergone a similar operation but without optic nerve injury would reveal genes specifically involved in responding to optic nerve injury (including repair), reducing detection of genes involved in the general stress and inflammatory responses. We discovered 120 genes were significantly (minimally two-fold with a P-value < or = 0.05) differentially expressed (up or down) at one or more time point. Among these was ATF3, a member of the cAMP-response element binding protein family. Work by others has indicated that elevated cAMP could be important in axon regeneration. We investigated ATF3 expression further by qRT-PCR, in situ hybridization and immunohistochemistry and found ATF3 expression is significantly upregulated in the ganglion cell layer of the retina, the nerve fiber layer and the optic nerve of the injured eye. The upregulation in retina is detectable by qRT-PCR by 24 h after injury, at which time ATF-3 mRNA levels are 8-fold higher than in retinas from sham-operated fish. We conclude ATF3 may be an important mediator of optic nerve regeneration-promoting gene expression in fish, a role which merits further investigation.

Diurnal and circadian retinomotor movements in zebrafish.

Key indicators of circadian regulation include the persistence of physiological rhythmicity in the absence of environmental time cues and entrainment of this rhythmicity by the ambient light cycle. In some teleosts, the inner segments of rod and cone photoreceptors contract and elongate according to changes in ambient lighting and the circadian cycle. Pigment granules in the retinal pigment epithelium (RPE) disperse and aggregate in a similar manner. Collectively, these movements are known as retinomotor movements. We report the histological characterization of diurnal and circadian retinomotor movements in zebrafish, Danio rerio. Adult fish subjected to a 14:10 light:dark (LD) cycle, constant darkness (DD), or constant light (LL) were sacrificed at 1-13 h intervals and processed for semithin sectioning of the retina. Using bright-field microscopy, 15 measurements of pigment granule position and the inner segment lengths of 30 rods and 30-45 cones were collected from the central third of the dorso-optic retina per time point. In LD, rods and cones followed a clear diurnal rhythm in their inner segment movements. Short-single, UV-sensitive cones were found to contract significantly 1 h before light onset in LD conditions. In DD conditions, the inner segments movements of short-single and double cones displayed statistically significant rhythms. RPE pigment granule movements are rhythmically regulated in both LD and DD although fluctuations are damped in the absence of photic cues. No significant retinomotor movements were observed in LL. These findings indicate retinomotor movements in zebrafish are differentially regulated by an endogenous oscillator and by light-dependent mechanisms.

Exogenous superoxide dismutase and catalase promote recovery of function in isolated rat heart after regional ischemia and may be transported from capillaries into myocytes

SummaryThe effects of infusing superoxide dismutase (SOD) and catalase (CAT) into the coronary circulation were investigated in isolated, working rat hearts prior to and during a 15 minute episode of regional ischemia followed by 30 minutes reperfusion. Aortic output, left ventricular pressure and dP/dT were recorded. Compared to untreated hearts, SOD and CAT significantly improved function during reperfusion, but had no effect during the pre-ischemic or the ischemic period. To investigate possible transport of SOD and CAT into rat myocytes, cryotome sections of isolated, Langendorff perfused rat hearts were exposed to rabbit antibody prepared against the exogenous SOD and CAT. Bound antibody was detected by the indirect-fluorescent antibody test. The interior of myocytes from rat hearts exposed to SOD and CAT bound antibodies prepared against these enzymes, whereas myocytes from rat hearts not exposed to exogenous SOD and CAT only bound the CAT antibodies. This indicates the anti-SOD we prepared is specific for exogenous SOD, and also suggests exogenous SOD can gain access to the cytoplasm of myocytes from the coronary circulation.

Discrete nuclear structures in actively growing neuroblastoma cells are revealed by antibodies raised against phosphorylated neurofilament proteins

BackgroundNuclear objects that have in common the property of being recognized by monoclonal antibodies specific for phosphoprotein epitopes and cytoplasmic intermediate filaments (in particular, SMI-31 and RT-97) have been reported in glial and neuronal cells, in situ and in vitro. Since neurofilament and glial filaments are generally considered to be restricted to the cytoplasm, we were interested in exploring the identity of the structures labeled in the nucleus as well as the conditions under which they could be found there.ResultsUsing confocal microscopy and western analysis techniques, we determined 1) the immunolabeled structures are truly within the nucleus; 2) the phosphoepitope labeled by SMI-31 and RT-97 is not specific to neurofilaments (NFs) and it can be identified on other intermediate filament proteins (IFs) in other cell types; and 3) there is a close relationship between DNA synthesis and the amount of nuclear staining by these antibodies thought to be specific for cytoplasmic proteins. Searches of protein data bases for putative phosphorylation motifs revealed that lamins, NF-H, and GFAP each contain a single tyrosine phosphorylation motif with nearly identical amino acid sequence.ConclusionWe therefore suggest that this sequence may be the epitope recognized by SMI-31 and RT-97 mABs, and that the nuclear structures previously reported and shown here are likely phosphorylated lamin intermediate filaments, while the cytoplasmic labeling revealed by the same mABs indicates phosphorylated NFs in neurons or GFAP in glia.

GFAP and nuclear lamins share an epitope recognized by monoclonal antibody J1-31

Monoclonal antibody J1-31 was raised against plaque materials taken from brains of patients who had suffered from multiple sclerosis (MS). Preliminary characterization of the antigen revealed it to be a protein of M(w) 68-70 kDa with both a cytoplasmic and nuclear localization. Here we report the results of isolation and peptide sequencing of the antigen from human brains, and immunocytochemical analysis of the antigen in F98 glioma cells. Purification and peptide sequencing indicate that the antibody recognizes a form of glial fibrillary acidic protein, possibly a phosphorylated variant. However, confocal immunocytochemistry and western analysis of F98 glioma cells raise the possibility that it also recognizes a phosphorylated epitope found in nuclear lamins. Analysis of the expression of the J1-31 epitope in F98 cells with respect to time in culture, cell density, and DNA synthesis showed a developmental relationship: cells that were engaged in rapid growth and DNA synthesis exhibited strong J1-31 staining in nuclei, whereas quiescent cells did not. We conclude that mAB J1-31 remains a useful antibody for studying multiple sclerosis, and is likely to prove useful in studies of the dynamics of nuclear lamins, particularly in models for wound-healing.

Intermediate filaments of zebrafish retinal and optic nerve astrocytes and Müller glia: differential distribution of cytokeratin and GFAP

BackgroundOptic nerve regeneration (ONR) following injury is a model for central nervous system regeneration. In zebrafish, ONR is rapid - neurites cross the lesion and enter the optic tectum within 7 days; in mammals regeneration does not take place unless astrocytic reactivity is suppressed. Glial fibrillary acidic protein (GFAP) is used as a marker for retinal and optic nerve astrocytes in both fish and mammals, even though it has long been known that astrocytes of optic nerves in many fish, including zebrafish, express cytokeratins and not GFAP. We used immunofluorescence to localize GFAP and cytokeratin in wild-type zebrafish and transgenic zebrafish expressing green fluorescent protein (GFP) under control of a GFAP promoter to determine the pattern of expression of intermediate filaments in retina and optic nerve.FindingsGFAP labeling and GFAP gene expression as indicated by GFP fluorescence was found only in the Müller glial cells of the retina. Within Müller cells, GFP fluorescence filled the entire cell while GFAP labelling was more restricted in distribution. No GFAP expression was observed in optic nerves. Cytokeratin labeling of astrocytes was observed throughout the optic nerve and less intensely in cells in the retinal inner plexiform layer. The retinal inner limiting membrane was strongly labeled by anti-cytokeratin.ConclusionsStudies of astrocyte function during ONR in zebrafish cannot solely rely on GFAP as an astrocyte marker or indicator of reactivity. Future studies of ONR in zebrafish should include evaluation of changes in cytokeratin expression and localization in the optic nerve.

Phosphorylated neurofilaments and SNAP-25 in cultured SH-SY5Y neuroblastoma cells

Components of the extracellular matrix (ECM) of mammals have profound effects on the behavior and differentiation of many different cell types. Here, we report the results of biochemical and immunocytochemical investigations of the expression of SNAP-25 and phosphorylated neurofilament proteins (NFs) by cells grown on coverslips, cells cultured in EHS-ECM gels, and cells in situ in rat brain. SNAP-25 and phosphorylated NFs were detected by immunofluorescence in all these environments but were not detectable by Western analysis in extracts of cells grown on coverslips. The results support the interpretation that EHS-ECM induces differentiation of SH-SY5Y cells in culture and suggest this system as a model system for study of nerve tissue formation and repair.

Inhibitors of adenosine catabolism improve recovery of dog myocardium after ischemia

SummaryThe effects of inhibitors of adenosine catabolism on contractile function and metabolites were assessed during 15 minutes of ischemia followed by 30 minutes of reperfusion in the open-chest dog heart. As compared to sham treatment, pretreatment with erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) and dipyridamole (DP) protected contractile function during ischemia, and improved recovery of high energy phosphate content and contractile fucntion during reperfusion following ischemia. Testing EHNA and DP in a free-radical generating system indicated both compounds have some scavenging ability, suggesting the effect of EHNA + DP may not be on adenosine nucleotide metabolism alone. Comparison of end diastolic segment lengths to contractile function indicated the results were not affected by changes in preload resulting from peripheral vasodilation.

Activating transcription factor 3 and reactive astrocytes following optic nerve injury in zebrafish

Nerve regeneration in the central nervous system is restricted in mammals, but fish and amphibians show amazing resiliency following injury to the central nervous system. We have examined the response of zebrafish (Danio rerio) to optic nerve injury to try to understand the differences between fish and mammals that enable fish to regenerate their optic nerves following crushing and severing. In previous work, we have shown that activating transcription factor 3 (atf3) is expressed at higher levels following optic nerve injury. Here we use a polyclonal anti-ATF3 antibody, anti-cytokeratin (KRT-18) and anti-bystin (BYSL) antibodies to show that Atf3 and Bysl colocalize with cytokeratin-expressing astrocytes in the optic nerve following severing. Furthermore, anti-ATF3 antibodies fail to colocalize with GFP in transgenic zebrafish expressing EGFP in astrocytes Tg(gfap:GFP) or oligodendrocytes Tg(olig2:EGFP). Interestingly, labeling of Atf3 was detected in retinal ganglion cell axons in both the nerve fiber layer and the optic nerve on the injured side. Finally, optic nerve astrocytes labeled with anti-bystin antibodies showed evidence of hypertrophy, suggesting that fish astrocytes in the optic nerve raise a bona fide reactive response to injury even though they do not express glial fibrillary acidic protein.