Impact of sociodemographic factors, stress, and communication on health-related quality of life in survivors of pediatric cancer.

BACKGROUND: While most research has largely focused on medical risks associated with reduced health-related quality of life (HRQOL) in survivors, sociodemographic and family factors may also play a role. Thus, we longitudinally examined sociodemographic factors and family factors associated with survivor HRQOL, including adolescent's cancer-specific stress, mother's general stress, and mother-adolescent communication. METHODS: Mothers (N = 80) and survivors (ages 10-23, N = 50) were assessed 5 years following initial diagnosis. Mothers completed measures regarding sociodemographic background adolescent's cancer-specific stress, mother's general stress, mother-adolescent communication, and adolescent HRQOL. Survivors also reported on their own HRQOL. Two hierarchical multiple regressions examined predictors of (a) mother's report of adolescent HRQOL, and (b) survivor's self-report of HRQOL. RESULTS: The final model predicting mother-reported adolescent HRQOL was significant, F(5,74) = 21.18, p < .001, and explained 59% of the variance in HRQoL. Significant predictors included adolescent stress (ß = -.37, p < .001), mothers' stress (ß = -.42, p < .001), and communication (ß = .19, p = .03). The final model predicting survivor-reported HRQOL was also significant, F(5,44) = 5.16, p < .01 and explained 24% of the variance in HRQOL. Significant predictors included adolescent stress (ß = -.37, p = .01) and communication (ß = -.31, p = .04). Sociodemographic factors were not a significant predictor of HRQOL in any model. CONCLUSION: Family stress and communication offer potential points of intervention to improve HRQOL of pediatric cancer survivors from mother and survivor perspectives. While additional research is needed, healthcare professionals should encourage stress management and strong mother-child communication to enhance survivors' long-term HRQOL. Such interventions may be complimentary to efforts targeting the known sociodemographic factors that often affect health.

Pilot Testing Transcreated Spanish-Language Study Materials for Symptom Research With Infants and Parents in the Neonatal Intensive Care Unit.

BACKGROUND: Rising admission rates of Hispanic/Latinx families to the neonatal intensive care unit (NICU) have increased the number of non-English-speaking individuals who may wish to participate in research studies. However, a lack of appropriately translated research study materials may limit the opportunity for these families to be involved in research that could impact the care that infants and families receive in the NICU. PURPOSE: The primary purpose was to pilot test study materials that were transcreated from English to Spanish with the assistance of a bilingual community advisory board with Spanish-speaking parents of NICU infants. METHODS: A total of 19 Spanish-speaking parents (15 mothers and 4 fathers) who were representative of the population of interest completed paper-and-pencil surveys, along with a cognitive interview. Preliminary data related to decision-making and goals of care, infant symptoms, and their experiences in the NICU were also collected. RESULTS: The internal reliability of the transcreated study instruments ranged from good to excellent (α= 0.82-0.99). Participants reported that study materials were not offensive and did not make them feel uncomfortable; however, they found some words/phrases to be confusing. Parents had the opportunity to provide suggested wording changes. IMPLICATIONS FOR PRACTICE AND RESEARCH: Language barriers and a lack of cultural responsiveness can affect the care that infants and their families receive. More accurate and culturally appropriate transcreation of study materials can remove barriers to research participation and facilitate better communication with non-English-speaking families, which may lead to the development of better-informed evidence-based interventions and clinical practices in the NICU.

Inclusion of Spanish-Speaking Families in NICU Symptom Research Using a Community Advisory Board.

BACKGROUND: Admission rates of Hispanic/Latinx families to the neonatal intensive care unit (NICU) are rising, yet knowledge regarding their experiences is limited. Non-English-speaking families are often excluded from research because study measures are not available in their language. The inclusion of these families in NICU research is crucial to understand the impact of cultural and language barriers in infant and family care, and the translation of study measures into Spanish could better ensure that these families' perspectives are included. However, previous research has utilized the standard translation-backtranslation method with which problems have been noted. PURPOSE: This article describes the planning, preparation, and action phases that were completed with the use of a community advisory board (CAB) to prepare and transcreate research study materials from English to Spanish for pilot testing. METHODS: Community members of interest were invited to form the CAB, and 2 meetings were held to review study materials in the English and Spanish versions. Prior to the second meeting, a professional translation company translated study materials from English to Spanish. RESULTS: Feedback from the CAB ensured consistent content and reading levels, and culturally appropriate language usage. The CAB also provided suggestions to the research protocol and advised sensitive methods of recruitment and measure administration. IMPLICATIONS FOR PRACTICE AND RESEARCH: More accurate and culturally appropriate transcreation of study measures can reduce barriers to research participation and facilitate better communication with non-English-speaking families in health equity research to better inform evidence-based interventions and clinical practices across diverse groups.

Communication within families about advanced pediatric cancer: A qualitative study.

OBJECTIVES: This qualitative study examined how families share information and feelings about advanced pediatric cancer from the perspective of both parents and children, as well as how these perspectives vary by child developmental stage. METHODS: Participants (24 mothers, 20 fathers, 23 youth [children and adolescents]) were from a larger longitudinal study at an academic pediatric hospital. Eligible youth had advanced cancer (physician-estimated prognosis of <60%, relapse, or refractory disease), were aged 5-19 years (>8 years old to participate independently), had an English-speaking parent, and lived within 140 miles of the hospital. Interviews were completed at enrollment and asked how families share information and emotions about the child's cancer as a family. RESULTS: Saturation was reached at 20 interviews for mothers, fathers, and youth. Analyses revealed 4 major themes: (A) parents managing cancer-related information based on child age/developmental stage and processing styles of family members; (B) parents withholding poor prognosis information and emotions to maintain positivity; (C) lack of personal and familial emotion sharing; and (D) emotion sharing among their family and externally. Both parents and youth endorsed themes A, C, and D, but only parents endorsed theme B. Adolescents endorsed more themes than children. Parents of children (as opposed to adolescents) endorsed theme A more. SIGNIFICANCE OF RESULTS: Although both parents and youth with advanced cancer were generally willing to talk about treatment, emotions were not consistently shared. Perspectives varied depending on the child's developmental stage. Clinicians should assess parent and child information and emotion-sharing needs and provide individualized support to families regarding communication about advanced cancer.

A method for quantifying pigment position in retinal pigment epithelium.

In wildtype mice, the pigment granules in the retinal pigment epithelium aggregate in the dark towards Bruch's membrane and disperse towards the photoreceptors in the light. We have developed a repeatable method amenable for quantifying pigment position in the RPE from wild type mice by estimating the population density of pigment granules, or pigment density, within 4 µm2 areas in the basal part of cells examined by transmission electron microscopy. To measure pigment position, 2 µm × 2 µm squares were aligned along the apical ends of the basal microvilli. The pigment granules within each 4 µm2 area were counted, and the average pigment density was calculated for each mouse. The average pigment density for light-adapted mice (n = 3 mice) was 1.3 pigment granules/µm2 (± 0.2 pigment granules/µm2). For dark-adapted wildtype mice (n = 3 mice), pigment density was 1.9 pigment granules/µm2 (± 0.3 pigment granules/µm2). Pigment density was statistically significantly different (p < 0.02) between light-adapted and dark-adapted mice, with pigment density higher in the dark-adapted mice. This method was implemented by four observers and their results were compared. No statistically significant differences were found in the measurements acquired by the different observers, illustrating the repeatability of the method.

Ammonia concentration at emergence and its effects on the recovery of different species of entomopathogenic nematodes.

The life cycle of entomopathogenic nematodes (EPN) occurs inside an insect cadaver and an accumulation of ammonia initiates as a consequence of the nematodes defecation. This accumulation reduces the food resources quality and creates a detrimental environment for nematodes. When a given ammonia concentration is reached, the nematodes start their emergence process, searching for a new host. In the present work, this parameter, ammonia triggering point (ATP) was measured in 7 Steinernema species/strains. The effect of different ammonia concentrations on the recovery process and their consequences in the nematodes survival were also investigated. The results indicate that ATP varies among nematode species; Steinernema glaseri showed the highest ATP of the evaluated species (1.98±2.6 mg of NH4-N*g of Galleria mellonella(-1)); whereas Steinernema riobrave presented the lowest ATP (1.16±0.1 mg of NH4-N*g of G. mellonella(-1)). On the other hand, the nematode emergence could be a repulsive response when ATP is reached. As the ammonia concentration increased the recovery percentage of Steinernema feltiae (Chile strain) dropped gradually from 79.4±11.9% in the control treatment to 0% when 1mg of NH4-N*ml of bacterial broth(-1) was added. It is possible, that emergence process could be a repulsive response of the nematodes due to ammonia concentration when is reaching the ATP. The role of ammonia inside the insect cadavers, might suggests connections with some stages of the EPN life cycle.

Characteristics of Critically Ill Infants at the End of Life in the Neonatal Intensive Care Unit.

Objectives: About 16,000 infants die in the neonatal intensive care unit (NICU) each year with many experiencing invasive medical treatments and high number of symptoms.1 To inform better management, we characterized diagnoses, symptoms, and patterns of care among infants who died in the NICU. Method: Retrospective electronic medical record (EMR) review of 476 infants who died following admission to a large regional level IV NICU in the United States over a 10-year period. Demographic, symptom, diagnosis, treatment, and end-of-life characteristics were extracted. Results: About half of infants were male (55.9%, n = 266), average gestational age was 31.3 weeks (standard deviation [SD] = 6.5), and average age at death was 40.1 days (SD = 84.5; median = 12; range: 0-835). Race was documented for 65% of infants, and most were White (67.0%). One-third of infants (n = 138) were seen by fetal medicine. Most infants experienced pain through both the month and week before death (79.6%), however, infants with necrotizing enterocolitis had more symptoms in the week before death. Based on EMR, infants had more symptoms, and received more medical interventions and comfort measures during the week before death compared with the month prior. Only 35% (n = 166) received a palliative care referral. Conclusions: Although the medical profiles of infants who die in the NICU are complex, the overall number of symptoms was less than in older pediatric populations. For infants at high risk of mortality rate, providers should assess for common symptoms over time. To manage symptoms as effectively as possible, both timely and continuous communication with parents and early referral to palliative care are recommended.

Associations between parental depression, communication, and self-worth of siblings bereaved by cancer.

A child's death from cancer may increase the risk for poor self-worth in bereaved siblings. Furthermore, bereaved parents may experience depressive symptoms and communicate differently with their surviving children. However, limited research has examined family factors associated with self-worth in bereaved siblings. Thus, we examined: (a) differences in parental depressive symptoms, parent-child communication, and sibling self-worth between bereaved and nonbereaved families and (b) indirect effects of parental depressive symptoms and communication quality on the association between bereavement and sibling self-worth. Bereaved parents and siblings were recruited 3-12 months after a child's death from cancer. Bereaved (n = 72) and nonbereaved families of classmates (n = 58) completed home-based questionnaires upon enrollment (T1), and 48 bereaved and 45 nonbereaved families completed 1-year follow-up (T2). Relative to controls at T1 and T2, bereaved mothers, but not fathers, reported more depressive symptoms. Bereaved siblings reported poorer maternal and similar paternal communication, and similar levels of self-worth compared to controls. Both cross-sectional and longitudinal serial mediation models for mothers were significant. Bereaved mothers were at greater risk for depressive symptoms, which adversely affected sibling self-worth over time through disrupted mother-child communication. The father sample was limited, but the cross-sectional model was nonsignificant. Mothers and fathers may grieve differently and may require different therapeutic approaches. Family-centered interventions should target bereaved mothers' emotional adjustment and communication to enhance sibling self-worth. Additionally, clinicians should bolster other sources of support for bereaved siblings to promote adaptive outcomes. (PsycInfo Database Record (c) 2023 APA, all rights reserved).

Comparative development and ocular histology between epigean and subterranean salamanders (Eurycea) from central Texas.

The salamander clade Eurycea from the karst regions of central Texas provides an ideal platform for comparing divergent nervous and sensory systems since some species exhibit extreme phenotypes thought to be associated with inhabiting a subterranean environment, including highly reduced eyes, while others retain an ancestral ocular phenotype appropriate for life above ground. We describe ocular morphology, comparing three salamander species representing two phenotypes-the surface-dwelling Barton Springs salamander (E. sosorum) and San Marcos salamander (E. nana) and the obligate subterranean Texas blind salamander (E. rathbuni) - in terms of structure and size of their eyes. Eyes were examined using confocal microscopy and measurements were made using ImageJ. Statistical analysis of data was carried out using R. We also provide a developmental series and track eye development and immunolocalization of Pax6 in E. sosorum and E. rathbuni. Adult histology of the surface-dwelling San Marcos salamander (E. nana) shows similarities to E. sosorum. The eyes of adults of the epigean species E. nana and E. sosorum appear fully developed with all the histological features of a fully functional eye. In contrast, the eyes of E. rathbuni adults have fewer layers, lack lenses and other features associated with vision as has been reported previously. However, in early developmental stages eye morphology did not differ significantly between E. rathbuni and E. sosorum. Parallel development is observed between the two phenotypes in terms of morphology; however, Pax6 labeling seems to decrease in the latter stages of development in E.rathbuni. We test for immunolabeling of the visual pigment proteins opsin and rhodopsin and observe immunolocalization around photoreceptor disks in E. nana and E. sosorum, but not in the subterranean E. rathbuni. Our results from examining developing salamanders suggest a combination of underdevelopment and degeneration contribute to the reduced eyes of adult E. rathbuni.

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.

Astrocytes as gate-keepers in optic nerve regeneration--a mini-review.

Animals that develop without extra-embryonic membranes (anamniotes--fish, amphibians) have impressive regenerative capacity, even to the extent of replacing entire limbs. In contrast, animals that develop within extra-embryonic membranes (amniotes--reptiles, birds, mammals) have limited capacity for regeneration as adults, particularly in the central nervous system (CNS). Much is known about the process of nerve development in fish and mammals and about regeneration after lesions in the CNS in fish and mammals. Because the retina of the eye and optic nerve are functionally part of the brain and are accessible in fish, frogs, and mice, optic nerve lesion and regeneration (ONR) has been extensively used as a model system for study of CNS nerve regeneration. When the optic nerve of a mouse is severed, the axons leading into the brain degenerate. Initially, the cut end of the axons on the proximal, eye-side of the injury sprout neurites which begin to grow into the lesion. Simultaneously, astrocytes of the optic nerve become activated to initiate wound repair as a first step in reestablishing the structural integrity of the optic nerve. This activation appears to initiate a cascade of molecular signals resulting in apoptotic cell death of the retinal ganglion cells axons of which make up the neural component of the optic nerve; regeneration fails and the injury is permanent. Evidence specifically implicating astrocytes comes from studies showing selective poisoning of astrocytes at the optic nerve lesion, along with activation of a gene whose product blocks apoptosis in retinal ganglion cells, creates conditions favorable to neurites sprouting from the cut proximal stump, growing through the lesion and into the distal portion of the injured nerve, eventually reaching appropriate targets in the brain. In anamniotes, astrocytes ostensibly present no such obstacle since optic nerve regeneration occurs without intervention; however, no systematic study of glial involvement has been done. In fish, vigorously growing neurites sprout from the cut axons and within a few days begin to re-enervate the brain. This review offers a new perspective on the role of glia, particularly astrocytes, as "gate-keepers;" i.e., as being permissive or inhibitory, by comparison between fish and mammals of glial function during ONR.

Duplicated Myosin V Genes in Teleosts Show Evolutionary Rate Variations among the Motor and Cargo-Binding Domains.

We analyzed evolutionary rates of conserved, duplicated myosin V (myo5) genes in nine teleost species to examine the outcomes of duplication events. Syntenic analysis and ancestral chromosome mapping suggest one tandem gene duplication event leading to the appearance of myo5a and myo5c, two rounds of whole genome duplication for vertebrates, and an additional round of whole genome duplication for teleosts account for the presence and location of the myo5 genes and their duplicates in teleosts and other vertebrates and the timing of the duplication events. Phylogenetic analyses reveal a previously unidentified myo5 clade that we refer to now as myo5bb. Analysis using dN/dS rate comparisons revealed large regions within duplicated myo5 genes that are highly conserved. Codons identified in other studies as encoding functionally important portions of the Myo5a and Myo5b proteins are shown to be highly conserved within the newly identified myo5bb clade and in other myo5 duplicates. As much as 30% of 319 codons encoding the cargo-binding domain in the myo5aa genes are conserved in all three codon positions in nine teleost species. For the myo5bb cargo-binding domain, 6.6% of 336 codons have zero substitutions in all nine teleost species. Using molecular evolution assays, we identify the myo5bb branch as being subject to evolutionary rate variation with the cargo-binding domain, having 20% of the sites under positive selection and the motor domain having 8% of its sites under positive selection. The high number of invariant codons coupled with relatively high dN/dS values in the region of the myo5 genes encoding the ATP-binding domain suggests the encoded proteins retain function and may have acquired novel functions associated with changes to the cargo-binding domain.

Carbachol-mediated pigment granule dispersion in retinal pigment epithelium requires Ca2+ and calcineurin.

BACKGROUND: Inside bluegill (Lepomis macrochirus) retinal pigment epithelial cells, pigment granules move in response to extracellular signals. During the process of aggregation, pigment motility is directed toward the cell nucleus; in dispersion, pigment is directed away from the nucleus and into long apical processes. A number of different chemicals have been found to initiate dispersion, and carbachol (an acetylcholine analog) is one example. Previous research indicates that the carbachol-receptor interaction activates a Gq-mediated pathway which is commonly linked to Ca2+ mobilization. The purpose of the present study was to test for involvement of calcium and to probe calcium-dependent mediators to reveal their role in carbachol-mediated dispersion. RESULTS: Carbachol-induced pigment granule dispersion was blocked by the calcium chelator BAPTA. In contrast, the calcium channel antagonist verapamil, and incubation in Ca2+-free medium failed to block carbachol-induced dispersion. The calcineurin inhibitor cypermethrin blocked carbachol-induced dispersion; whereas, two protein kinase C inhibitors (staurosporine and bisindolylmaleimide II) failed to block carbachol-induced dispersion, and the protein kinase C activator phorbol 12-myristate 13-acetate failed to elicit dispersion. CONCLUSION: A rise in intracellular calcium is necessary for carbachol-induced dispersion; however, the Ca2+ requirement is not dependent on extracellular sources, implying that intracellular stores are sufficient to enable pigment granule dispersion to occur. Calcineurin is a likely Ca2+-dependent mediator involved in the signal cascade. Although the pathway leads to the generation of diacylglycerol and calcium (both required for the activation of certain PKC isoforms), our evidence does not support a significant role for PKC.

Determining Zebrafish Epitope Reactivity to Commercially Available Antibodies.

Antibodies raised against mammalian proteins may exhibit cross-reactivity with zebrafish proteins, making these antibodies useful for fish studies. However, zebrafish may express multiple paralogues of similar sequence and size, making them difficult to distinguish by traditional Western blot analysis. To identify the zebrafish proteins that are recognized by an antimammalian antibody, we developed a system to screen putative epitopes by cloning the sequences between the yeast SUMO protein and a C-terminal 6xHis tag. The recombinant fusion protein was expressed in Escherichia coli and analyzed by Western blot to conclusively identify epitopes that exhibit cross-reactivity with the antibodies of interest. This approach can be used to determine the species cross-reactivity and epitope specificity of a wide variety of peptide antigen-derived antibodies.

Uptake of 3H-cAMP by retinal pigment epithelium isolated from bluegill sunfish (Lepomis macrochirus).

BACKGROUND: In bluegill sunfish, the melanin-containing pigment granules of the retinal pigment epithelium undergo cyclic movements in response both to ambient lighting and circadian cues. Pigment granules aggregate into the cell body at night (in the dark), and disperse into apical processes during the day (in the light). Regulation of pigment granule aggregation in a number of fishes depends on modulating the intracellular levels of cyclic adenosine monophosphate. RESULTS: Here we show isolated RPE takes up cyclic adenosine monophosphate (cAMP) in a saturable manner, exogenously applied cAMP induces pigment granule aggregation in retinal pigment epithelium isolated from bluegill, and aggregation induced in this manner is inhibited by treatment with probenecid, an organic anion transport inhibitor. CONCLUSION: Our results raise the possibility that cAMP functions as a messenger secreted from the neural retina to signal darkness to the RPE, which takes it up. It further suggests that organic anion transport systems are the route by which cAMP crosses RPE cell membranes since probenecid inhibits extracellular cAMP from causing pigment granule aggregation.

Activation of muscarinic acetylcholine receptors elicits pigment granule dispersion in retinal pigment epithelium isolated from bluegill.

BACKGROUND: In fish, melanin pigment granules in the retinal pigment epithelium disperse into apical projections as part of the suite of responses the eye makes to bright light conditions. This pigment granule dispersion serves to reduce photobleaching and occurs in response to neurochemicals secreted by the retina. Previous work has shown that acetylcholine may be involved in inducing light-adaptive pigment dispersion. Acetylcholine receptors are of two main types, nicotinic and muscarinic. Muscarinic receptors are in the G-protein coupled receptor superfamily, and five different muscarinic receptors have been molecularly cloned in human. These receptors are coupled to adenylyl cyclase, calcium mobilization and ion channel activation. To determine the receptor pathway involved in eliciting pigment granule migration, we isolated retinal pigment epithelium from bluegill and subjected it to a battery of cholinergic agents. RESULTS: The general cholinergic agonist carbachol induces pigment granule dispersion in isolated retinal pigment epithelium. Carbachol-induced pigment granule dispersion is blocked by the muscarinic antagonist atropine, by the M1 antagonist pirenzepine, and by the M3 antagonist 4-DAMP. Pigment granule dispersion was also induced by the M1 agonist 4-[N-(4-chlorophenyl) carbamoyloxy]-4-pent-2-ammonium iodide. In contrast the M2 antagonist AF-DX 116 and the M4 antagonist tropicamide failed to block carbachol-induced dispersion, and the M2 agonist arecaidine but-2-ynyl ester tosylate failed to elicit dispersion. CONCLUSIONS: Our results suggest that carbachol-mediated pigment granule dispersion occurs through the activation of Modd muscarinic receptors, which in other systems couple to phosphoinositide hydrolysis and elevation of intracellular calcium. This conclusion must be corroborated by molecular studies, but suggests Ca2+-dependent pathways may be involved in light-adaptive pigment dispersion.

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

BACKGROUND: Nuclear 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. RESULTS: Using 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. CONCLUSION: We 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.

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.

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.