Expression of mRNA encoding extracellular matrix glycoproteins SPARC and SC1 is temporally and spatially regulated in the developing cochlea of the rat inner ear.

SPARC is a multifunctional extracellular matrix (ECM) glycoprotein that shares partial sequence homology with SC1/hevin. These ECM molecules exhibit calcium-binding properties and modulate cellular interactions. This study examines the expression of SC1 and SPARC mRNA in the developing cochlea of the rat inner ear prior to and after the onset of hearing. At all ages examined, SC1 mRNA is highly expressed in neurons of the spiral ganglion. In contrast, SPARC transcripts are not detected in the spiral ganglion but are enriched in the temporal bone and cartilaginous otic capsule surrounding the cochlea. Both SC1 and SPARC mRNA are expressed in connective tissue elements involved in maintaining ionic homeostasis of cochlear fluids. SC1 mRNA is localized to type III fibrocytes of the spiral ligament (slg) and marginal cells of the stria vascularis, while SPARC mRNA is apparent in the spiral limbus and type I fibrocytes of the slg. At postnatal day 10, SPARC mRNA shows a dramatic change in expression. High levels of SPARC transcripts are induced in Deiters cells (dc) of the organ of Corti. Interestingly, this induction of SPARC mRNA correlates with the onset of hearing. This suggests that SPARC may play a role in calcium regulation in dc when functional maturation of the cochlea is attained and rapid changes in calcium levels are required.

Immunocytochemical localization of HrpA and HrpZ supports a role for the Hrp pilus in the transfer of effector proteins from Pseudomonas syringae pv. tomato across the host plant cell wall.

The Hrp pilus, composed of HrpA subunits, is an essential component of the type III secretion system in Pseudomonas syringae. We used electron microscopy (EM) and immunocytochemistry to examine production of the pilus in vitro from P. syringae pv. tomato strain DC3000 grown under hrp-inducing conditions on EM grids. Pili, when labeled with antibodies to HrpA, developed rapidly in a nonpolar manner shortly after the detection of the hrpA transcript and extended up to 5 microm into surrounding media. Structures at the base of the pilus were clearly differentiated from the basal bodies of flagella. The HrpZ protein, also secreted via the type III system, was found by immunogold labeling to be associated with the pilus in vitro. Accumulation and secretion of HrpA and HrpZ were also examined quantitatively after the inoculation of wild-type DC3000 and hrpA and hrpZ mutants into leaves of Arabidopsis thaliana. The functional pilus crossed the plant cell wall to generate tracks of immunogold labeling for HrpA and HrpZ. Mutants that produced HrpA but did not assemble pili were nonpathogenic, did not secrete HrpA protein, and were compromised for the accumulation of HrpZ. A model is proposed in which the rapidly elongating Hrp pilus acts as a moving conveyor, facilitating transfer of effector proteins from bacteria to the plant cytoplasm across the formidable barrier of the plant cell wall.

Differential mRNA expression of the related extracellular matrix glycoproteins SC1 and SPARC in the rat embryonic nervous system and skeletal structure.

SPARC is a multifunctional extracellular matrix glycoprotein that shares partial sequence homology with SC1. These extracellular matrix molecules are thought to play important roles in modulating cellular interactions. In vitro, SPARC has been shown to exhibit anti-adhesive activity. In the present investigation, in situ hybridization is used to compare the expression patterns of SC1 and SPARC mRNA in the rat embryo. Results show that SC1 and SPARC expression is spatially and temporally regulated. SC1 mRNA is strongly expressed in the embryonic brain and spinal cord, whereas SPARC mRNA is enriched in craniofacial cartilage and skeletal structures. This differential expression pattern in the rat embryo suggests that SC1 plays an important role in the developing nervous system, whereas SPARC participates primarily in events associated with skeletal development. However at embryonic day 17, SC1 and SPARC mRNA show parallel expression patterns in areas of the cerebellum undergoing cell migratory events.

A loss of resistance to avirulent bacterial pathogens in tobacco is associated with the attenuation of a salicylic acid-potentiated oxidative burst.

The role of salicylic acid (SA) in events occurring before cell death during the hypersensitive reaction (HR) was investigated in leaves of wild-type tobacco Samsun NN and in transgenic lines expressing salicylate hydroxylase (35S-SH-L). Challenge of 35S-SH-L tobacco with avirulent strains of Pseudomonas syringae gave rise to symptoms resembling those normally associated with a compatible response to virulent strains in terms of visible phenotype, kinetics of bacterial multiplication, and escape from the infection site. Compared with responses in wild-type tobacco, both the onset of plant cell death and the induction of an active oxygen species-responsive promoter (AoPR1-GUS) were delayed following challenge of 35S-SH-L plants with avirulent bacteria. The oxidative burst occurring after challenge with avirulent bacteria was visualized histochemically and quantified in situ. H2O2 accumulation at reaction sites was evident within 1 h after inoculation in wild-type tobacco, whereas in 35S-SH-L plants the onset of H2O2 accumulation was delayed by 2-3 h. The delay in H2O2 generation was correlated with a reduction in the transient rise in SA that usually occurred within 1-2 h following inoculation in wild-type plants. Our data indicate that an early transient rise in SA potentiates the oxidative burst, with resultant effects on accumulation of H2O2, plant cell death and also defence-gene induction, factors that together may determine the outcome of plant-pathogen interactions.

Heat shock transcription factors and the hsp70 induction response in brain and kidney of the hyperthermic rat during postnatal development.

Heat shock transcription factor (HSF) 1 levels increase in brain regions and decline in kidney during postnatal rat development. In both neonatal and adult rats, levels of HSF1 protein in brain and kidney are proportional to the levels of HSF DNA-binding activity and the magnitude of heat shock protein hsp70 induction after thermal stress. There appears to be more HSF1 protein in adult brain than is needed for induction of hsp70 after thermal stress, suggesting that HSF1 may have other functions in addition to its role as a stress-inducible activator of heat shock genes. HSF2 protein levels decline during postnatal rat development in brain regions and kidney. Gel mobility shift analysis shows that HSF2 is not in a DNA-binding form in the neonatal brain and kidney, suggesting that HSF2 may not be involved in the constitutive expression of hsps in early postnatal development. There is no apparent relationship between levels of HSF2 protein and basal levels of hsp90, hsp70, heat shock cognate protein hsc70, and hsp60.

Selective transport of SC1 mRNA, encoding a putative extracellular matrix glycoprotein, during postnatal development of the rat cerebellum and retina.

The selective transport of mRNA species into peripheral processes of cells is an important aspect of gene expression in the nervous system. In this study, we report the transport of SC1 mRNA into the distal processes of Bergmann glial (BG) cells at particular stages of development. SC1 is a putative anti-adhesive extracellular matrix (ECM) glycoprotein that is expressed not only in the developing central nervous system (CNS) but also in the adult brain. The intracellular distribution of SC1 mRNA was examined in two highly laminated neural structures, the cerebellum and retina, during postnatal development and in the adult rat. Our results indicate that SC1 mRNA expression is both spatially and temporally regulated. SC1 message was localized to BG cell bodies at postnatal day 5 (P5) and P10. However, by P15 through to the adult, SC1 mRNA was transported to distal processes of BG cells in the synapse-rich molecular layer (ML) of the cerebellum. In the developing rat retina, SC1 mRNA was expressed in specific neuronal populations by P10, however, transport of SC1 message to the dendrites of these retinal neurons was not detected during development or in the adult. These results indicate neural mechanisms which control the timing and cell type in which selective transport of SC1 mRNA is observed. The localization of SC1 mRNA to the distal processes of BG cells in the synapse-rich ML of the cerebellum could facilitate local control of SC1 protein synthesis which may play roles in synapse formation during development and in synaptic plasticity in the adult.

Heat shock proteins Hsp27 and Hsp32 localize to synaptic sites in the rat cerebellum following hyperthermia.

Stressful stimuli activate the heat shock (stress) response in which a set of heat shock proteins (hsps) is induced, which play roles in cellular repair and protective mechanisms. Most studies in the mammalian nervous system have focused on Hsp70, however, the present investigation targets other members of the induced set, namely Hsp27 and Hsp32. In response to hyperthermia, these hsps are strongly induced in Bergmann glial cells in the rat brain and transported into their radial fibers, which project into the 'synaptic-enriched' molecular layer of the cerebellum. Using subcellular fractionation and immunoelectron microscopy, hyperthermia-induced Hsp27 and Hsp32 were detected in synaptic elements and in perisynaptic glial processes. These results suggest that stress-induced Hsp27 and Hsp32 may contribute to repair and protective mechanisms at the synapse.

Localization of the heat-shock protein Hsp70 to the synapse following hyperthermic stress in the brain.

Heat-shock proteins are induced in response to cellular stress. Although heat-shock proteins are known to function in repair and protective mechanisms, their relationship to critical neural processes, such as synaptic function, has received little attention. Here we investigate whether the major heat-shock protein Hsp70 localizes to the synapse following a physiologically relevant increase in temperature in the mammalian nervous system. Our results indicate that hyperthermia-induced Hsp70 is associated with pre- and postsynaptic elements, including the postsynaptic density. The positioning of Hsp70 at the synapse could facilitate the repair of stress-induced damage to synaptic proteins and also contribute to neuroprotective events at the synapse.

Induction of SC1 mRNA encoding a brain extracellular matrix glycoprotein related to SPARC following lesioning of the adult rat forebrain.

SC1 is an extracellular matrix (ECM) glycoprotein related to SPARC which exhibits anti-adhesive properties. ECM molecules are thought to play important roles in influencing cell shape, proliferation and migration during neurogenesis. Following localized injury to the adult rat forebrain, a biphasic induction of SC1 mRNA was apparent, namely a rapid, transient induction at 1 day post-lesion in cortical neurons which border the lesion site followed by a more prolonged induction in astrocytes which are proximal to the wound site. A similar SC1 induction pattern was observed in the hippocampus in response to the injury. SPARC mRNA exhibits a divergent pattern of induction because it is induced in mature blood vessels close to the lesion and in blood vessels which develop following the trauma. Thus mRNAs encoding the related ECM glycoproteins SC1 and SPARC are induced in different cell populations in the adult forebrain during the neural response to localized injury.

Sex difference in the relationship of calcium and magnesium excretion to glycaemic control in type 1 diabetes mellitus.

In order to assess the variability and possible causes of calcium and magnesium losses in diabetes mellitus, urinary calcium and magnesium excretion were monitored six monthly over a 3-year period in 108 stable, type 1 diabetic patients who were having assessment of their clinical status and glycaemic control over the same period. In the patients studied the ranges of excretion of both calcium and magnesium were considerably wider than our non-diabetic reference ranges but the within subject variation in excretion was high. However, using mean values obtained over the study period, a direct relationship was observed between the excretion of both calcium and magnesium and HbA1 in female patients (P < 0.01) but not in males who had similar HbA1 values. The urinary excretion of calcium and magnesium did not relate to any of the other clinical or biochemical indices measured, including body mass index, daily insulin dose, retinal status or albumin excretion. It is suggested that, in poorly controlled patients, females may have a greater risk than males of developing the complications associated with chronic calcium and magnesium loss.

Neuroprotection at Drosophila synapses conferred by prior heat shock.

Synapses are critical sites of information transfer in the nervous system, and it is important that their functionality be maintained under stressful conditions to prevent communication breakdown. Here we show that synaptic transmission at the Drosophila larval neuromuscular junction is protected by prior exposure to heat shock that strongly induces expression of heat shock proteins, in particular hsp70. Using a macropatch electrode to record synaptic activity at individual, visualized boutons, we found that prior heat shock sustains synaptic performance at high test temperatures through pre- and postsynaptic alterations. After heat shock, nerve impulses release more quantal units at high temperatures and exhibit fewer failures of release (presynaptic modification), whereas the amplitude of quantal currents remains more constant than does that in nonheat-shocked controls (postsynaptic modification). The time course of these physiological changes is similar to that of elevated hsp70. Thus, stress-induced neuroprotective mechanisms maintain function at synapses by modifying their properties.

Cellular localization of the heat shock transcription factors HSF1 and HSF2 in the rat brain during postnatal development and following hyperthermia.

The heat shock transcription factor HSF1 mediates the induction of heat shock genes in response to temperature elevation and other traumatic events. The induced hsps play roles in cellular repair and protective mechanisms. Immunocytochemistry revealed that in the unstressed rat, HSF1 was already prepositioned in the nucleus at abundant levels in both neuronal and glial cell types. Following a fever-like temperature, glial cells rapidly induced hsp70 whereas populations of large neurons did not. The lack of hsp70 induction in these neurons in vivo did not appear to be due to deficiencies in levels of nuclear HSF1. During postnatal development of the cerebellum, levels of HSF1 increased progressively from day 1 to 30. Members of the hsp gene set are also constitutively expressed in the unstressed animal and play roles as molecular chaperones. HSF2, which has been proposed as a developmental regulator of constitutive heat shock gene expression, demonstrated a developmental alteration in cellular localization, namely a nuclear distribution in neurons at postnatal day 2 and a cytoplasmic localization at day 30. During postnatal development the overall levels of neural HSF2 declined. This profile showed no obvious correlation with previously observed levels of constitutive hsp expression during postnatal neural development.

Characterization of the heat shock response in the gills of sea lampreys and a brook lamprey at different intervals of their life cycles.

The heat shock response (HSR) was characterized in the gills of two lamprey species that differ with respect to their adult life history. In vivo labelling with [35S]methionine revealed an enhanced synthesis of heat shock proteins (HSPs) having approximate molecular weights of 70 kDa (HSP70) and 90 kDa (HSP90) following heat treatment. Induction of the HSR occurred in larval lampreys (ammocoetes) following temperature elevations of 13-16 degrees C for the parasitic species, the sea lamprey (Petromyzon marinus) and 16-20 degrees C for the nonparasitic species, the brook lamprey (Lampetra appendix). The case in L. appendix represents the greatest increase in temperature required to induce the HSR in gill tissue among aquatic poikilotherms studied to data and induction occurs within a temperature range (25-29 degrees C) not normally experienced by these animals. Western blotting detected the presence of 70 and 90 kDa HSPs and HSP70 levels were greater in post-metamorphic L. appendix than in ammocoetes both before and after heat shock. The HSR of lampreys appears to be induced during times of emergency when large, rapid temperature increases are experienced. The high set-point temperature for induction of the response may be a consequence of both the environments they presently inhabit and their experiences during evolution.

Constitutive expression of heat shock proteins Hsp90, Hsc70, Hsp70 and Hsp60 in neural and non-neural tissues of the rat during postnatal development.

Heat shock proteins (Hsps) are a group of highly conserved proteins, that are constitutively expressed in most cells under normal physiological conditions. Previous work from our laboratory has shown that neurons in the adult brain exhibit high levels of Hsp90 and Hsc70 mRNA and protein, as well as basal levels of Hsp70 mRNA. We have now investigated the expression of Hsp90, Hsc70, Hsp60 and Hsp70 in neural and non-neural tissues of the rat during postnatal development, a time of extensive cell differentiation. Western blot analysis revealed constitutive expression of these Hsps early in postnatal development. Developmental profiles of these Hsps suggest that they are differentially regulated during postnatal development of the rat. For example, while levels of Hsp90 decrease somewhat in certain developing brain regions, levels of Hsp60 show a developmental increase, and Hsc70 protein is abundant throughout postnatal neural development. Low basal levels of Hsp70 are also observed in the developing and adult brain. A pronounced decrease in Hsp90 and Hsc70 was observed during postnatal development of the kidney while levels of Hsp60 increased. In addition, tissue-specific differences in the relative levels of these Hsps between brain and non-brain regions were found. Immunocytochemical studies demonstrated a neuronal localization of Hsp90, Hsc70 and Hsp60 at all stages of postnatal development examined as well as in the adult, suggesting a role for Hsps in both the developing and fully differentiated neuron. The developmental expression of subunit IV of cytochrome oxidase was similar to that of Hsp60, a protein localized predominantly to mitochondria.

Expression of SC1 is associated with the migration of myotomes along the dermomyotome during somitogenesis in early mouse embryos.

SC1 is a secreted glycoprotein with a high amino acid sequence similarity to SPARC (Secreted Protein, Acidic, Rich in Cysteine). SC1 transcripts were first detected in mouse embryos after day 8.5 post coitus (p.c.) in somites at the medial lip of the dermomyotome. Expression of SC1 transcripts by the progenitor cells continued as they began involuting under the dermomyotome and during their migration along the lateral wall of the dermomyotome. After myotome migration was completed, SC1 mRNA expression was downregulated in the trunk region. The data indicate that SC1 expression is restricted to the initial stages of epaxial myotome differentiation and migration, undergoing rapid downregulation prior to myotome emigration from the somitic environment.

SPARC/osteonectin mRNA is induced in blood vessels following injury to the adult rat cerebral cortex.

Recently we described the pattern of expression of the anti-adhesive glycoprotein SPARC/osteonectin in the developing and adult brain. SPARC mRNA was present in developing blood vessels during neurogenesis, but was not detected in the mature vasculature. We have now examined the effect of a lesion to the adult rat cerebral cortex on the expression of SPARC by in situ hybridization. SPARC mRNA was increased in the zone proximal to the wound at 3 to 10 days after cortical brain injury. During this period, SPARC was induced in mature blood vessels close to the lesion site and in blood vessels which develop following injury. These results suggest a role for SPARC in the process of angiogenesis following injury to the adult cerebral cortex.

Effect of hyperthermia on the transcription rate of heat-shock genes in the rabbit cerebellum and retina assayed by nuclear run-ons.

The induction of heat-shock protein 70 (hsp70) mRNA in the hyperthermic rabbit brain has been examined previously by using Northern blotting and in situ hybridization procedures that measure steady-state levels of mRNA, which may be influenced by transcript stability and transcription rate. In the present investigation, the in vivo transcription rate of hsp70 has been examined by using run-on transcription assays on isolated brain nuclei. A major up-regulation in the transcription rate of hsp70 was observed between 0.75 and 1.50 hours after hyperthermia in the cerebellum and the retina. Gel-mobility shift assays revealed that the time course of conversion of heat-shock transcription factor (HSF1) to a DNA-binding form paralleled the transcriptional induction profile of hsp70. The transcription rates of several nonheat-shock genes were also studied in the hyperthermic brain, and little change was noted relative to the induction of hsp70. Thus, a physiologically relevant increase in temperature of 2.5 degrees C induces a major up-regulation in the in vivo transcription rate of hsp70 in the nervous system with little affect on the transcription rates of other genes.

Developmental analysis of factors binding to the mouse 68-kDa neurofilament promoter.

Whole tissue extracts prepared from mouse brain regions at various postnatal ages were characterized for binding of factors to the DNase I hypersensitive site (HSSI) which is located closest to the transcription start site of the 68-kDa mouse neurofilament gene (NF-L). Gel mobility shift assays detected changes in factor binding during postnatal development of the neocortex. Competition experiments suggested that one of the complexes resulted from factor binding to a 9 bp sequence found in both the light and medium neurofilament promoter regions (NF-L/M). Gel mobility shifts performed with an oligonucleotide probe containing the NF-L/M sequence detected two brain-specific DNA-protein complexes, and a third complex in both brain and liver. During cerebellar and neocortical development, one of the NF-L/M complexes was most intense at postnatal day 10 when transcription of the NF-L gene is upregulated.

Differential induction of heat shock mRNA in oligodendrocytes, microglia, and astrocytes following hyperthermia.

A time course analysis of hsp70 mRNA induction in response to a physiologically relevant increase in body temperature of 2.6 degrees C was performed in the rabbit forebrain. A protocol that combined in situ hybridization and cytochemistry on the same tissue section was employed to identify reactive glial cell types. Cytochemical markers for astrocytes, microglia, and oligodendrocytes were utilized in combination with a DIG-labelled hsp70 riboprobe, which permitted mRNA localization at high resolution. Four glial cell body-enriched regions of the rabbit forebrain were examined, namely, cortical layer 1, hippocampal fissure, corpus callosum, and fimbria. Maximal hsp70 mRNA induction was observed in 2 and 3 h hyperthermic animals. The colocalization analysis demonstrated that hsp70 mRNA was induced in oligodendrocytes and microglia, but not in forebrain GFAP positive astrocytes. In addition, cell counts were performed which showed that almost all oligodendrocytes induced hsp70 mRNA while a subpopulation of microglial cells responded. These data are consistent with the notion that oligodendrocytes, microglia, and astrocytes exhibit distinct thresholds for activation of the heat shock response following a physiologically relevant increase in body temperature.