Shared medical appointments for patients with a nondiabetic physical chronic illness: A systematic review.

OBJECTIVES: Shared medical appointments are group appointments, with an optional individual consultation, for patients diagnosed with chronic illnesses. Shared medical appointments improve diabetes management, but little is known about their use for other illnesses. The objective was to determine the effect that shared medical appointments have on patients with a physical chronic illness, healthcare providers, and the healthcare system. METHODS: A systematic review was conducted searching databases from January 1970 to September 2016. Eligible trials evaluated shared medical appointments for patients with a homogeneous chronic illness, excluding diabetes and mental illness. Screening, data extraction, and risk of bias were conducted independently by two authors. Analysis was descriptive. RESULTS: Of 2364 citations, nine randomized trials were included. Shared medical appointments were evaluated for cardiovascular illnesses (four studies), breast cancer, chronic kidney disease, Parkinson's disease, stress urinary incontinence, and carpal tunnel syndrome. Compared to usual care, no negative effects on patient quality of life, knowledge and satisfaction were reported. One study reported no difference in healthcare provider satisfaction. Another study showed fewer hospital admissions for patients who attended shared medical appointments. DISCUSSION: Few rigorous studies evaluated the use of shared medical appointments for chronic illnesses. Overall, there appears to be no patient harms. Further studies should include more objective outcomes and larger sample sizes.

Usp9X Regulates Cell Death in Malignant Peripheral Nerve Sheath Tumors.

Malignant peripheral nerve sheath tumors (MPNSTs) are the leading cause of death in neurofibromatosis type 1 (NF1) patients. Current treatment modalities have been largely unsuccessful in improving MPNST patient survival, making the identification of new therapeutic targets urgent. In this study, we found that interference with Usp9X, a deubiquitinating enzyme which is overexpressed in nervous system tumors, or Mcl-1, an anti-apoptotic member of the Bcl-2 family whose degradation is regulated by Usp9X, causes rapid death in human MPNST cell lines. Although both Usp9X and Mcl-1 knockdown elicited some features of apoptosis, broad spectrum caspase inhibition was ineffective in preventing knockdown-induced MPNST cell death suggesting that caspase-independent death pathways were also activated. Ultrastructural examination of MPNST cells following either Usp9X interference or pharmacological inhibition showed extensive cytoplasmic vacuolization and swelling of endoplasmic reticulum (ER) and mitochondria most consistent with paraptotic cell death. Finally, the Usp9X pharmacological inhibitor WP1130 significantly reduced human MPNST growth and induced tumor cell death in an in vivo xenograft model. In total, these findings indicate that Usp9X and Mcl-1 play significant roles in maintaining human MPNST cell viability and that pharmacological inhibition of Usp9X deubiquitinase activity could be a therapeutic target for MPNST treatment.

Symptom practice guide for telephone assessment of patients with cancer treatment-related cardiotoxic dyspnea: Adaptation and evaluation of acceptability.

BACKGROUND: Patients with cancer treatment-related cardiotoxicity, which may manifest as heart failure (HF), can present with dyspnea. Nurses frequently assess, triage and offer self-care strategies to patients experiencing dyspnea in both the cardiology and oncology settings. However, there are no known tools available for nurses to manage patients in the setting of cancer treatment-related cardiotoxicity. The objective of this study was to adapt and evaluate the acceptability of an evidence-informed symptom practice guide (SPG) for use by nurses over the telephone for the assessment, triage, and management of patients experiencing dyspnea due to cancer treatment-related cardiotoxicity. METHODS: The CAN-IMPLEMENT© methodology guided this descriptive study. A systematic search was conducted in four databases to identify cardio-oncology and HF guidelines and systematic reviews. Screening was conducted by two reviewers, with data extracted into a recommendation matrix from eligible guidelines and systematic reviews on: assessment criteria, medications, and/or self-care strategies to manage dyspnea. Healthcare professionals with an expertise in oncology and/or cardiology were recruited using purposeful and snowball sampling. Evaluation of acceptability of the adapted SPG was gathered through semi-structured interviews and a survey with open- and closed-ended questions. Quantitative findings and participant feedback from the interviews and the open-ended survey questions were analyzed descriptively. RESULTS: Of 490 citations, seven HF guidelines were identified. Evidence from these guidelines was added to the original SPG. Eleven healthcare professionals completed the interview and acceptability survey. The adapted SPG was iteratively revised three times during the interviews. The original SPG was adaptable, and participants indicated the adapted SPG was comprehensive, easy to follow, and would be useful in clinical practice. CONCLUSIONS: This study highlights the lack of knowledge tools and available clinical practice guidelines to guide healthcare professionals to assess, triage and/or offer self-care strategies to patients with cancer treatment-related cardiotoxic dyspnea. Moreover, most nurses require assistance to differentiate among the various causes of dyspnea from oncology treatment in order to triage severity appropriately. Further research should focus on evaluating the validity of the adapted SPG in clinical practice.

Overexpression of GAB2 in ovarian cancer cells promotes tumor growth and angiogenesis by upregulating chemokine expression.

We previously found that the scaffold adapter GRB2-associated binding protein 2 (GAB2) is amplified and overexpressed in a subset of primary high-grade serous ovarian cancers and cell lines. Ovarian cancer cells overexpressing GAB2 are dependent on GAB2 for activation of the phosphatidylinositol 3-kinase (PI3K) pathway and are sensitive to PI3K inhibition. In this study, we show an important role of GAB2 overexpression in promoting tumor angiogenesis by upregulating expression of multiple chemokines. Specifically, we found that suppression of GAB2 by inducible small hairpin RNA in ovarian cancer cells inhibited tumor cell proliferation, angiogenesis and peritoneal tumor growth in immunodeficient mice. Overexpression of GAB2 upregulated the secretion of several chemokines from ovarian cancer cells, including CXCL1, CXCL2 and CXCL8. The secreted chemokines not only signal through endothelial CXCR2 receptor in a paracrine manner to promote endothelial tube formation, but also act as autocrine growth factors for GAB2-induced transformation of fallopian tube secretory epithelial cells and clonogenic growth of ovarian cancer cells overexpressing GAB2. Pharmacological inhibition of inhibitor of nuclear factor kappa-B kinase subunit ß (IKKß), but not PI3K, mechanistic target of rapamycin (mTOR) or mitogen-activated protein kinase (MEK), could effectively suppress GAB2-induced chemokine expression. Inhibition of IKKß augmented the efficacy of PI3K/mTOR inhibition in suppressing clonogenic growth of ovarian cancer cells with GAB2 overexpression. Taken together, these findings suggest that overexpression of GAB2 in ovarian cancer cells promotes tumor growth and angiogenesis by upregulating expression of CXCL1, CXCL2 and CXCL8 that is IKKß-dependent. Co-targeting IKKß and PI3K pathways downstream of GAB2 might be a promising therapeutic strategy for ovarian cancer that overexpresses GAB2.

Assessment of oncolytic HSV efficacy following increased entry-receptor expression in malignant peripheral nerve sheath tumor cell lines.

Limited expression and distribution of nectin-1, the major herpes simplex virus (HSV) type-1 entry-receptor, within tumors has been proposed as an impediment to oncolytic HSV (oHSV) therapy. To determine whether resistance to oHSVs in malignant peripheral nerve sheath tumors (MPNSTs) was explained by this hypothesis, nectin-1 expression and oHSV viral yields were assessed in a panel of MPNST cell lines using γ134.5-attenuated (Δγ134.5) oHSVs and a γ134.5 wild-type (wt) virus for comparison. Although there was a correlation between nectin-1 levels and viral yields with the wt virus (R=0.75, P =0.03), there was no correlation for Δγ134.5 viruses (G207, R7020 or C101) and a modest trend for the second-generation oHSV C134 (R=0.62, P=0.10). Nectin-1 overexpression in resistant MPNST cell lines did not improve Δγ134.5 oHSV output. While multistep replication assays showed that nectin-1 overexpression improved Δγ134.5 oHSV cell-to-cell spread, it did not confer a sensitive phenotype to resistant cells. Finally, oHSV yields were not improved with increased nectin-1 in vivo. We conclude that nectin-1 expression is not the primary obstacle of productive infection for Δγ134.5 oHSVs in MPNST cell lines. In contrast, viruses that are competent in their ability to counter the antiviral response may derive benefit with higher nectin-1 expression.

Knockout of plasminogen activator inhibitor 1 gene reduces amyloid beta peptide burden in a mouse model of Alzheimer's disease.

Accumulation of amyloid beta peptide (Aß) in the brain is a pathological hallmark of Alzheimer's disease (AD); the underlying mechanism, however, is not well understood. In this study, we show that expression of plasminogen activator inhibitor 1 (PAI-1), a physiological inhibitor of tissue type and urokinase type plasminogen activators (tPA and uPA), increases with age in the brain of wild type and Aß precursor protein-presenilin 1 (APP/PS1) transgenic mice as well as in AD patients. Most importantly, we show that knocking out the PAI-1 gene dramatically reduces Aß burden in the brain of APP/PS1 mice but has no effect on the levels of full-length APP, alpha or beta C-terminal fragments. Furthermore, we show that knocking out the PAI-1 gene leads to increases in the activities of tPA and plasmin, and the plasmin activity inversely correlates with the amounts of SDS insoluble Aß40 and Aß42. Together, these data suggest that increased PAI-1 expression/activity contributes importantly to Aß accumulation during aging and in AD probably by inhibiting plasminogen activation and thus Aß degradation.

Phylogenetic and functional biomakers as indicators of bacterial community responses to mixed-waste contamination.

Few studies have demonstrated changes in community structure along a contaminant plume in terms of phylogenetic, functional, and geochemical changes, and such studies are essential to understand how a microbial ecosystem responds to perturbations. Clonal libraries of multiple genes (SSU rDNA, nirK, nirS, amoA, pmoA, and dsrAB) were analyzed from groundwater samples (n = 6) that varied in contaminant levels, and 107 geochemical parameters were measured. Principal components analyses (PCA) were used to compare the relationships among the sites with respect to the biomarker (n = 785 for all sequences) distributions and the geochemical variables. A major portion of the geochemical variance measured among the samples could be accounted for by tetrachloroethene, 99Tc, No3, SO4, Al, and Th. The PCA based on the distribution of unique biomarkers resulted in different groupings compared to the geochemical analysis, but when the SSU rRNA gene libraries were directly compared (deltaC(xy) values) the sites were clustered in a similar fashion compared to geochemical measures. The PCA based upon functional gene distributions each predicted different relationships among the sites, and comparisons of Euclidean distances based upon diversity indices for all functional genes (n = 432) grouped the sites by extreme or intermediate contaminant levels. The data suggested that the sites with low and high perturbations were functionally more similar than sites with intermediate conditions, and perhaps captured the overall community structure better than a single phylogenetic biomarker. Moreover, even though the background site was phylogenetically and geochemically distinct from the acidic sites, the extreme conditions of the acidic samples might be more analogous to the limiting nutrient conditions of the background site. An understanding of microbial community-level responses within an ecological framework would provide better insight for restoration strategies at contaminated field sites.

Changes in bacterial community structure correlate with initial operating conditions of a field-scale denitrifying fluidized bed reactor.

High levels of nitrate are present in groundwater migrating from the former waste disposal ponds at the Y-12 National Security Complex in Oak Ridge, TN. A field-scale denitrifying fluidized bed reactor (FBR) was designed, constructed, and operated with ethanol as an electron donor for the removal of nitrate. After inoculation, biofilms developed on the granular activated carbon particles. Changes in the bacterial community of the FBR were evaluated with clone libraries (n = 500 partial sequences) of the small-subunit rRNA gene for samples taken over a 4-month start-up period. Early phases of start-up operation were characterized by a period of selection, followed by low diversity and predominance by Azoarcus-like sequences. Possible explanations were high pH and nutrient limitations. After amelioration of these conditions, diversification increased rapidly, with the appearance of Dechloromonas, Pseudomonas, and Hydrogenophaga sequences. Changes in NO3, SO4, and pH also likely contributed to shifts in community composition. The detection of sulfate-reducing-bacteria-like sequences closely related to Desulfovibrio and Desulfuromonas in the FBR have important implications for downstream applications at the field site.

Neuregulin-1beta modulates in vivo entorhinal-hippocampal synaptic transmission in adult rats.

Neuregulin-1 (NRG-1) proteins and their erbB receptors are essential for neuronal development during embryogenesis and may contribute importantly to neuronal function in the adult brain. This study tests the hypothesis that NRG-1beta acts as a modulator of synaptic activity in the adult brain, specifically at hippocampal formation synapses. Adult, male Sprague-Dawley rats were anesthetized and a recording electrode with an attached stainless steel microinjector was stereotaxically positioned to record field potentials (fEPSP) in either the dentate gyrus or the cornu ammonis (CA) 1 field of the hippocampus. The entorhinal cortex was continuously stimulated via a paired stainless steel electrode. Microinjection of NRG-1beta significantly increased the slope of the fEPSP in the dentate gyrus in a dose-dependent manner. Compared with a low dose (20 nM), a high dose (100 nM) of NRG-1beta induced a shorter latency response that was of greater magnitude. Responses to NRG-1beta were abolished by pretreatment with a selective, reversible erbB tyrosine kinase inhibitor, PD158780 (100 microM). Further, PD158780 (100 microM) itself significantly decreased the entorhinal-dentate fESPS slope by about 15%. Neither equimolar (100 nM) nor hypermolar (100 microM) sucrose or heat-inactivated NRG-1beta (100 nM) significantly altered the entorhinal-dentate fEPSP slope. In contrast to its effect at the entorhinal-dentate synapse, NRG-1beta (100 nM) depressed, and PD158780 potentiated entorhinal-CA1 synaptic transmission. Thus, in adult rats NRG-1beta potentiates transmission at the entorhinal-dentate synapse but suppresses transmission at the entorhinal-CA1 synapse. These observations indicate that NRG-1 is not only a developmental growth factor, but also modifies synaptic transmission in adult rat brain.

Targeting and functional role of N-RAP, a nebulin-related LIM protein, during myofibril assembly in cultured chick cardiomyocytes.

Targeting and functional effects of N-RAP domains were studied by expression as GFP-tagged fusion proteins in cultured embryonic chick cardiomyocytes. GFP-tagged N-RAP was targeted to myofibril precursors, myofibril ends and cell contacts, expression patterns that are similar to endogenous N-RAP. The GFP-tagged N-RAP LIM domain (GFP-N-RAP-LIM) was targeted to the membrane in cells with myofibril precursors and cell-cell contacts. The GFP-tagged super repeats (N-RAP-SR) and the GFP-tagged domain normally found in between the super repeats and the LIM domain (N-RAP-IB) were each observed at sites of myofibril assembly, incorporating into myofibril precursors in a manner similar to full length N-RAP. However, unlike full-length N-RAP, N-RAP-SR and N-RAP-IB were also found in mature myofibrils, associating with the sarcomeric actin filaments and the Z-lines, respectively. N-RAP-IB was also colocalized with alpha-actinin at cell contacts. Each of the N-RAP constructs could inhibit the formation of mature myofibrils in cultured cardiomyocytes, with the effects of N-RAP-SR and N-RAP-IB depending on the time of transfection. The results show that each region of N-RAP is crucial for myofibril assembly. Combining the targeting and functional effects of N-RAP domains with information in the literature, we propose a new model for initiation of myofibrillogenesis.

ErbB transmembrane tyrosine kinase receptors are differentially expressed throughout the adult rat central nervous system.

The neuregulin (NRG) family of growth and differentiation factors and their erbB receptors contribute importantly to the development of the nervous system, but their distribution and function in the adult brain are poorly understood. The present study showed that erbB2, erbB3, and erbB4 transcripts and protein are distributed throughout all areas of adult rat brain. These three receptors were differentially expressed in neurons and glia. Some neurons expressed only a subset of erbB kinases, whereas other neurons expressed all three erbB receptors but sequestered each of these polypeptides into distinct cellular compartments. In synapse-rich regions, erbB immunoreactivity appeared as punctate-, axon-, and/or dendrite-associated staining, suggesting that NRGs are involved in the formation and maintenance of synapses in adult brain. ErbB labeling also was present in neuronal soma, indicating that NRGs act at sites in addition to the synapse. Glia in adult brain also differentially expressed erbB3 and erbB4. Approximately half of the erbB3 labeling in white matter was associated with S100beta+/glial fibrillary acidic protein negative macroglia (i.e., oligodendrocytes or glial fibrillary acidic protein negative astrocytes). In contrast, macroglia in gray matter did not express erbB3. The remaining erbB3 immunoreactivity in white matter and erbB4 glial staining seemed to be associated with microglia. These results showed that erbB receptors are expressed widely in adult rat brain and that each erbB receptor subtype has a distinct distribution. The differential distributions of erbB receptors in neurons and glia and the known functional differences between these kinases suggest that NRGs have distinct effects on these cells. The continued expression of NRGs and their erbB receptors in mature brain also implies that these molecules perform important functions in the brain throughout life.

Glutamate transporter EAAT2 splice variants occur not only in ALS, but also in AD and controls.

OBJECTIVE: To ascertain the specificity of alternatively spliced mRNA variants of the astroglial glutamate transporter EAAT2 for ALS. BACKGROUND: An important hypothesis for ALS pathogenesis is that motor neuron injury may result from chronically elevated glutamate levels in the CNS. Supporting this idea are reports of decreased glutamate transport in ALS. This in turn has recently been suggested to be due to the presence of aberrant mRNA splice variants for EAAT2 in ALS. METHODS: Postmortem human brain tissue was obtained from different brain regions of patients with ALS, normal controls (NC), and patients with AD and Lewy body dementia (LB)-neurodegenerative diseases in which motor neurons are unaffected. Brain RNA was analyzed for EAAT2 isoforms using reverse transcription PCR and cDNA cloning/sequencing methods. RESULTS: Splice variants lacking exons 7 or 9 were present in ALS brain, as previously reported, but were also present in brains from NC, AD, and LB patients. PCR product sequence analyses from non-ALS brain show variant splicing identical to that reported for ALS. Quantitative PCR analysis shows that these isoforms may be somewhat more abundant in ALS than AD, LB, and NC brains. CONCLUSIONS: EAAT2 mRNA splice variants are found in the brains of NC and AD patients, as in ALS. The authors cannot exclude the possibility that quantitative changes in variant EAAT2 isoforms might relate directly, or indirectly, to ALS pathology. However, the qualitative presence of these "abnormal" EAAT2 splice variants does not appear to be sufficient to explain motor neuron degeneration in ALS.

Attenuation of experimental autoimmune demyelination in complement-deficient mice.

The exact mechanisms leading to CNS inflammation and myelin destruction in multiple sclerosis and in its animal model, experimental allergic encephalomyelitis (EAE) remain equivocal. In both multiple sclerosis and EAE, complement activation is thought to play a pivotal role by recruiting inflammatory cells, increasing myelin phagocytosis by macrophages, and exerting direct cytotoxic effects through the deposition of the membrane attack complex on oligodendrocytes. Despite this assumption, attempts to evaluate complement's contribution to autoimmune demyelination in vivo have been limited by the lack of nontoxic and/or nonimmunogenic complement inhibitors. In this report, we used mice deficient in either C3 or factor B to clarify the role of the complement system in an Ab-independent model of EAE. Both types of complement-deficient mice presented with a markedly reduced disease severity. Although induction of EAE led to inflammatory changes in the meninges and perivascular spaces of both wild-type and complement-deficient animals, in both C3(-/-) and factor B(-/-) mice there was little infiltration of the parenchyma by macrophages and T cells. In addition, compared with their wild-type littermates, the CNS of both C3(-/-) and factor B(-/-) mice induced for EAE are protected from demyelination. These results suggest that complement might be a target for the therapeutic treatment of inflammatory demyelinating diseases of the CNS.

Myofibrillogenesis and formation of cell contacts mediate the localization of N-RAP in cultured chick cardiomyocytes.

The expression of N-RAP was investigated in immuofluorescently stained embryonic chick cardiomyocyte cultures. After 1 day in culture, the cardiomyocytes were spherical and N-RAP, titin, alpha-actinin, and vinculin were all diffusely distributed. As the cardiomyocytes spread and formed myofibrils and cell contacts, N-RAP became localized to distinct areas in the cells. During myofibrillogenesis, N-RAP was found concentrated in premyofibrils. As the premyofibrils transformed into bundles of mature myofibrils, N-RAP became concentrated at the longitundal ends of the cells, and was not found in the mature sarcomeres. At sites of cell-cell contacts, N-RAP was localized to the cell junction even in cells without any significant myofibril formation. As the cell-cell contacts became more extensive and formed structures resembling the intercalated disks found in hearts, N-RAP became even more specifically concentrated at these junctions. The results show that myofibrillogenesis and cell contact formation can each independently target N-RAP to the longitudinal ends of cardiomyocytes.

Expression of JE (monocyte chemoattractant protein-1) is induced by sciatic axotomy in wild type rodents but not in C57BL/Wld(s) mice.

Recruitment of hematogenous myelomonocytic cells into injured peripheral nerve is essential for axonal regeneration. The monocyte chemoattractant protein-1 (JE) and melanoma growth stimulatory activity/gro (KC) "immediate early" gene products may be important in this process as these proteins are potent chemoattractants for macrophages and neutrophils, respectively. To test this hypothesis, we examined JE and KC activation in rat sciatic nerve 0-30 days after surgical transection. RT-PCR and in situ hybridization analyses of JE and KC expression demonstrates these mRNAs are present in injured nerve, first being expressed by a cellular subpopulation within the zone of trauma by 1.5 hours after injury. By 16 hours posttransection a subpopulation of JE-positive endoneurial cells is found in the proximal stump and throughout the distal nerve segment, with maximal mRNA accumulation occurring 1 day after injury and expression persisting to 18 days postaxotomy, a period preceding and coincident with macrophage infiltration. In contrast, by 3 days postaxotomy KC expression is markedly diminished, consistent with the limited neutrophilic response to nerve injury. JE expression was also examined in C57BL/Wld(s) mice, which have delayed Wallerian degeneration associated with a failure of macrophage recruitment, and their parental C57BL/6J strain. Although JE mRNA is inducible in sciatic nerve from C57BL/6J mice, these transcripts are undetectable in injured nerve from C57BL/Wld(s) mice. Our findings suggest that activation of the JE locus is at least partially responsible for macrophage invasion of injured peripheral nerve. Furthermore, defective postaxotomy macrophage recruitment in C57BL/Wld(s) mice may involve a failure of JE induction.

Neurotrophin sensitivity of prevertebral and paravertebral rat sympathetic autonomic ganglia.

Prevertebral and paravertebral sympathetic autonomic ganglia respond differently to a large number of experimental and clinical insults. The selective involvement of subpopulations of sympathetic neurons may reflect differences in their response to neurotrophic substances. To test this hypothesis, we investigated the response of prevertebral and paravertebral rat sympathetic ganglia to selected neurotrophic substances in vivo and in vitro and identified the ganglionic distribution of neurons expressing high affinity neurotrophin receptor mRNAs. Dissociated cultures of embryonic prevertebral and paravertebral ganglionic neurons showed comparable responses to NGF deprivation and only small differences in their response to rescue with other trophic substances. In situ hybridization studies of adult rat sympathetic ganglia using probes specific for the high-affinity neurotrophin receptor transcripts trks A, B, and C demonstrated that neurons in both prevertebral and paravertebral sympathetic ganglia express predominantly trkA receptors in vivo. In addition, increased tyrosine hydroxylase (TOH) activity was induced only by doses of neurotrophic substances that activate trkA and showed only small differences between neonatal prevertebral and paravertebral ganglia. Although small differences in the sensitivity of pre- and paravertebral sympathetic neurons to various neurotrophins have been identified in our studies, they are unlikely, in isolation, to explain major differences in the sensitivity of these ganglia to neuropathologic processes.

Calcium transients and calcium homeostasis in adult mouse fast-twitch skeletal muscle fibers in culture.

Skeletal muscle fibers enzymatically dissociated from adult mouse flexor digitorum brevis muscles were maintained in culture for up to 8 days. After various times in culture, fibers were loaded with fura 2, and Ca2+ transients for trains of 1, 5, and 10 action potentials (100 Hz) triggered by external electrical stimulation were calculated from fluorescence ratio records corrected for noninstantaneous reaction of fura 2 with Ca2+. The decay rate constants of Ca2+ transients decreased with increasing stimulation duration, indicating a slowing of the Ca(2+)-removal properties with increased stimulation duration. After 6 days in culture, Ca2+ decay rate constants decreased dramatically for all stimulation durations and the differences in decay rate constants among 1, 5, and 10 pulses became smaller. Intracellular parvalbumin content measured by single-fiber immunofluorescence decreased with time in culture in parallel with the decrease in the decay rate constant of Ca2+ transients. Our results suggest that there is a correlation between parvalbumin content and the decay rate constant of the Ca2+ transient.

Decay of calcium transients after electrical stimulation in rat fast- and slow-twitch skeletal muscle fibres.

1. Calcium transients were calculated from fura-2 fluorescence signals (corrected for kinetic delays in the Ca(2+)-fura-2 reaction) from single rat skeletal muscle fibres, either fully dissociated from the fast-twitch flexor digitorum brevis (FDB) muscle or in small bundles from the slow-twitch soleus muscle. Fibres or bundles were embedded in agarose gel to inhibit movement and stimulated by single or trains of 1-2 ms electrical pulses (100 Hz, 2-400 ms train duration). 2. The rate constant of decay of [Ca2+] determined from single-exponential fits to the final decay phase of [Ca2+] after a single action potential was considerably faster in FDB fibres than in soleus fibres. As the stimulation duration increased, the rate constant of [Ca2+] decay decreased for both the FDB and soleus fibres, but the effect was greater in FDB than in soleus fibres. 3. Using the magnitude of the decline in the rate constant of [Ca2+] decay with increasing stimulation duration as an index of relative contribution of the saturable Ca2+ binding sites on parvalbumin, subpopulations termed 'high', 'medium' and 'low', referring to estimated parvalbumin content, were determined within each group of FDB and soleus fibres. In fibres assigned to the 'high' and 'medium' groups, parvalbumin was the major contributor (50-73%) to the [Ca2+] decay rate constant after a single action potential. In fibres in the 'low' group, parvalbumin contributed only 0-28% to the rate constant of [Ca2+] decay. 4. Fluorescence recordings using mag-fura-2, a lower-affinity Ca2+ indicator expected to be in equilibrium with myoplasmic Ca2+, gave similar values for both the [Ca2+] decay rate constant after a single action potential and the decrease in this rate constant with increased stimulation duration, as found for the fura-2 [Ca2+] transients from FDB and soleus fibres. Thus, the observed differences in decay rate of Ca2+ were not introduced by kinetic correction of the fura-2 recordings, but are attributed to differences in the Ca2+ binding and transport properties of fast- and slow-twitch mammalian fibres.

Expression of neuregulins and their putative receptors, ErbB2 and ErbB3, is induced during Wallerian degeneration.

Schwann cell dedifferentiation and proliferation is a prerequisite to axonal regeneration in the injured peripheral nervous system. The neuregulin (NRG) family of growth and differentiation factors may play a particularly important role in this process, because these axon-associated molecules are potent Schwann cell mitogens and differentiation factors in vitro. We have examined Schwann cell DNA synthesis and the expression of NRGs and their receptors, the erbB membrane tyrosine kinases, in rat sciatic nerve, sensory ganglia, and spinal cord 0-30 d postaxotomy. Analysis of NRG cDNAs from these tissues revealed several novel splice variants and showed that cells endogenous to injured nerve express NRG mRNAs. A selective induction of mRNAs encoding the glial growth factor (GGF) subfamily of NRGs occurs in nerve beginning 3 d postaxotomy and thus coincides with the onset of Schwann cell DNA synthesis. In later stages of Wallerian degeneration, however, Schwann cell mitogenesis markedly decreases, whereas elevated GGF expression persists. Of the four known erbB kinases, Schwann cells express both erbB2 and erbB3 receptors over the entire interval studied. Expression of erbB2 and erbB3 is coordinately induced in response to axotomy, indicating that Schwann cell responses to NRGs may be modulated by changes in receptor density. Neuregulin (including transmembrane precursors) and erbB protein are associated with Schwann cells postaxotomy. Thus, in contrast to the concept of NRGs as axon-associated mitogens, our findings suggest that NRGs produced by Schwann cells themselves may be partially responsible for Schwann cell proliferation during Wallerian degeneration, probably acting via autocrine or paracrine mechanisms.