Reduction in re-rupture rates following implementation of return-to-sport testing after anterior cruciate ligament reconstruction in 313 patients with a mean follow-up of 50 months.

OBJECTIVES: The objective of this study was to assess the mid-term effectiveness of a return to sport (RTS) test in relation to preventing anterior cruciate ligament (ACL) re-rupture and contralateral ACL injury following ACL reconstruction (ACLR). Furthermore, this study aimed to assess the timing of passing a, RTS-test after surgery, and the effect age has on RTS outcomes. METHODS: Patients undergoing ACLR between August 2014 and December 2018 took an RTS-test following rehabilitation. The RTS-test consisted of the Anterior Cruciate Ligament Return to Sport After Injury Scale, a single-leg hop, a single-leg triple hop, a single-leg triple cross-over hop, a box-drop vertical jump down, a single-leg 4-rep max-incline leg press, and a modified agility T test. RTS-passing criteria were ≥90% limb symmetry index in addition to defined takeoff and landing parameters. Mid-term review assessed sporting level, ACL re-injury, and contralateral ACL injury. RESULTS: A total of 352 patients underwent RTS-testing, following ACLR with 313 (89%) contactable at follow-up, a mean of 50 months (standard deviation: 11.41, range: 28-76) after surgery. The re-rupture rate was 6.6% after passing the RTS-test and 10.3% following failure (p â€‹= â€‹0.24), representing a 36% reduction. Contralateral ACL injury rate after surgery was 6% and was 19% lower in those passing the RTS test. The mean age of patients passing their first RTS-test was significantly higher than that of those who failed (p â€‹= â€‹0.0027). Re-ruptures in those who passed the RTS test first time occurred late (>34 months), compared to those who failed first time, which all occurred early (<33 months) (p â€‹= â€‹0.0015). The mean age of re-rupture was significantly less than those who did not sustain a re-rupture (p â€‹= â€‹0.025). CONCLUSION: Passing a RTS-test following ACLR reduces ACL re-rupture by 36.21% and contralateral ACL injury by 19.15% at mid-term follow-up. Younger patients are more likely to fail a RTS-test and are at higher risk of contralateral ACL rupture.

Prenatal Evaluation of Intracranial Hemorrhage on Fetal MRI: A Retrospective Review.

BACKGROUND AND PURPOSE: The evaluation and characterization of germinal matrix hemorrhages have been predominantly described on postnatal head sonography in premature neonates. However, germinal matrix hemorrhages that are seen in premature neonates can be also seen in fetuses of the same postconceptual age and are now more frequently encountered in the era of fetal MR imaging. Our aim was to examine and describe the MR imaging findings of fetuses with intracranial hemorrhage. MATERIALS AND METHODS: A retrospective review of diagnostic-quality fetal MRIs showing intracranial hemorrhage from January 2004 to May 2020 was performed. Images were reviewed by 2 radiologists, and imaging characteristics of fetal intracranial hemorrhages were documented. Corresponding postnatal imaging and clinical parameters were reviewed. RESULTS: One hundred seventy-seven fetuses with a mean gestational age of 25.73 (SD, 5.01) weeks were included. Germinal matrix hemorrhage was identified in 60.5% (107/177) and nongerminal matrix hemorrhage in 39.5% (70/177) of patients. Significantly increased ventricular size correlated with higher germinal matrix hemorrhage grade (P < .001). Fetal growth restriction was present in 21.3% (20/94) of our population, and there was no significant correlation with germinal matrix grade or type of intracranial hemorrhage. An increased incidence of neonatal death with grade III germinal matrix hemorrhages (P = .069) compared with other grades was identified; 23.2% (16/69) of the neonates required ventriculoperitoneal shunts, with an increased incidence in the nongerminal matrix hemorrhage group (P = .026). CONCLUSIONS: MR imaging has become a key tool in the diagnosis and characterization of intracranial hemorrhage in the fetus. Appropriate characterization is important for optimizing work-up, therapeutic approach, and prenatal counseling.

Plant defense guilds.

Optimal plant defense should incorporate any mechanisms that influence the feeding behavior of potential pests. From a diverse collection of examples suggesting that the defense of a plant may be improved in the company of specific neighbors, we discuss a framework of operational mechanisms that begin to clarify some aspects of the recognized influence of species diversity on herbivory. Neighbors serve as insectary plants for herbivore predators and parasites, and influence herbivore feeding behavior by repelling, masking, attracting, and decoying. Insectary plants lower the numerical response of herbivores by increasing the efficiency of their predators and parasites. Repellent plants primarily lower functional response by causing the predator to fail to locate or reject its normal prey. Attractant-decoy plants dilute herbivore impact by drawing off herbivores, either increasing or decreasing their numerical and functional response (or either). The concept of gene conservation guilds adds diversionary and delaying tactics to the adaptation-counteradaptation view of plant-herbivore coevolution. The useful life of a given gene for resistance may best be extended by mechanisms that disrupt genetic tracking (specialization) by herbivores. Some plants may remain inedible not because their chemistry or morphology represents an evolutionary impasse, but because they live in an environment that provides acceptable options of variable quality. Feeding environments that provide little or no choice promote specialization by forcing physiological adaptation. Conversely, the evolutionary momentum of specializing herbivores may be lowered by enhancing their susceptibility, either by selection against virulent individuals, or by decreasing the exposure frequency of susceptible genotypes. The latter mechanism of conserving susceptible individuals takes advantage of herbivore behavioral sensitivity to variable plant quality. Direct selection against virulent genotypes requires temporal cycling of the herbivore population between resistant and nonresistant hosts. Both events may occur within defense guilds that provide acceptable feeding options of similar but distinctive quality.

Mite-plant associations from the eocene of southern australia.

Acarodomatia or "mite houses" are located on leaves of many present-day angiosperms and are inhabited by mites that may maintain leaf hygiene. Eocene deposits in southern Australia have yielded acarodomatia on fossil leaves of Elaeocarpaceae and Lauraceae and also contain oribatid mites with close affinities to those that inhabit the acarodomatia of the closest living relatives of the fossil plant taxa. The data indicate that mite-plant associations may have been widespread in southern Australia 40 million years ago.

Cell-specific regulation of agrin RNA splicing in the chick ciliary ganglion.

Alternative splicing results in production of four agrin proteins (agrin0, agrin8, agrin11, and agrin19) with different AChR aggregating activities. However, the cellular origin of mRNAs encoding each agrin isoform remains unknown. Using single-cell PCR, we demonstrate that in the chick ciliary ganglion, nonneuronal cells express only mRNA encoding agrin0, whereas neurons express one or any combination of agrin mRNAs. Moreover, significant differences were observed between the agrin mRNA profiles of ciliary and choroid neurons in the ganglion. The abundance of each agrin mRNA, the fraction of neurons expressing each transcript, and the combinations of transcripts expressed by neurons also change during development. Our results demonstrate that transcripts encoding agrin proteins with high AChR aggregating activity are expressed exclusively by neurons in the ciliary ganglion and that alternative splicing of agrin mRNA is regulated during development and in a cell-specific manner.

Alterations in the expression and gating of Drosophila sodium channels by mutations in the para gene.

Mutations in the para gene specifically affect the expression of sodium currents in Drosophila. While 65% of wild-type embryonic neurons in culture express sodium currents, three distinct mutations in the para locus resulted in a decrease in the fraction of cells from which sodium currents could be recorded. This reduction was allele-dependent: macroscopic sodium currents were exhibited in 49% of the neurons in parats1 cultures, 35% in parats2, and only 2% in paraST76. Voltage-clamp experiments demonstrated that the parats2 mutation also affected the gating properties of sodium channels. These results provide convincing evidence that para, a gene recently shown to exhibit sequence similarity to vertebrate sodium channels alpha subunits, encodes functional sodium channels in Drosophila. The finding that one para allele (paraST76) can virtually eliminate the expression of sodium currents strongly argues that the para gene codes for the majority of sodium channels in cultured embryonic neurons.

Mutant ubiquitin found in Alzheimer's disease causes neuritic beading of mitochondria in association with neuronal degeneration.

A dinucleotide deletion in human ubiquitin (Ub) B messenger RNA leads to formation of polyubiquitin (UbB)+1, which has been implicated in neuronal cell death in Alzheimer's and other neurodegenerative diseases. Previous studies demonstrate that UbB+1 protein causes proteasome dysfunction. However, the molecular mechanism of UbB+1-mediated neuronal degeneration remains unknown. We now report that UbB+1 causes neuritic beading, impairment of mitochondrial movements, mitochondrial stress and neuronal degeneration in primary neurons. Transfection of UbB+1 induced a buildup of mitochondria in neurites and dysregulation of mitochondrial motor proteins, in particular, through detachment of P74, the dynein intermediate chain, from mitochondria and decreased mitochondria-microtubule interactions. Altered distribution of mitochondria was associated with activation of both the mitochondrial stress and p53 cell death pathways. These results support the hypothesis that neuritic clogging of mitochondria by UbB+1 triggers a cascade of events characterized by local activation of mitochondrial stress followed by global cell death. Furthermore, UbB+1 small interfering RNA efficiently blocked expression of UbB+1 protein, attenuated neuritic beading and preserved cellular morphology, suggesting a potential neuroprotective strategy for certain neurodegenerative disorders.

Evidence that glucocorticoid- and cyclic AMP-induced apoptotic pathways in lymphocytes share distal events.

WEHI7.2 murine lymphocytes undergo apoptotic death when exposed to glucocorticoids or elevated levels of intracellular cyclic AMP (cAMP), and these pathways are initiated by the glucocorticoid receptor (GR) and protein kinase A, respectively. We report the isolation and characterization of a novel WEHI7.2 variant cell line, WR256, which was selected in a single step for growth in the presence of dexamethasone and arose at a frequency of approximately 10(-10). The defect was not GR-related, as WR256 expressed functional GR and underwent GR-dependent events associated with apoptosis, such as hormone-dependent gene transcription and inhibition of cell proliferation. Moreover, the glucocorticoid-resistant phenotype was stable in culture and did not revert after treatment with 5-azacytidine or upon stable expression of GR cDNA. In addition, WR256 did not exhibit the diminished mitochondrial activity commonly associated with apoptosis. Interestingly, WR256 was also found to be resistant to 8-bromo-cAMP and forskolin despite having normal levels of protein kinase A activity and the ability to induce cAMP-dependent transcription. We examined the steady-state transcript levels of bcl-2, a gene whose protein product acts dominantly to inhibit thymocyte apoptosis, to determine whether elevated bcl-2 expression could account for the resistant phenotype. Our data showed that bcl-2 RNA levels were similar in the two cell lines and not altered by either dexamethasone or 8-bromo-cAMP treatment. These results suggest that WR256 exhibits a "deathless" phenotype and has a unique defect in a step of the apoptotic cascade that may be common to the glucocorticoid- and cAMP-mediated cell death pathways.

Retinoid X receptors stimulate and 9-cis retinoic acid inhibits 1,25-dihydroxyvitamin D3-activated expression of the rat osteocalcin gene.

The vitamin D receptor (VDR) binds the vitamin D-responsive element (VDRE) as a heterodimer with an unidentified receptor auxiliary factor (RAF) present in mammalian cell nuclear extracts. VDR also interacts with the retinoid X receptors (RXRs), implying that RAF may be related to the RXRs. Here we demonstrate that highly purified HeLa cell RAF contained RXR beta immunoreactivity and that both activities copurified and precisely coeluted in high-resolution hydroxylapatite chromatography. Furthermore, an RXR beta-specific antibody disrupted VDR-RAF-VDRE complexes in mobility shift assays. These data strongly indicate that HeLa RAF is highly related to or is identical to RXR beta. Consequently, the effect of the 9-cis retinoic acid ligand for RXRs was examined in 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]-activated gene expression systems. Increasing concentrations of 9-cis retinoic acid (1 nM to 1 microM) markedly reduced 1,25(OH)2D3-dependent accumulation of osteocalcin mRNA in osteoblast-like ROS 17/2.8 cells. All-trans retinoic acid also interfered with vitamin D responsiveness, but it was consistently less potent than the 9-cis isomer. Transient transfection studies revealed that attenuation by 9-cis retinoic acid was at the transcriptional level and was mediated through interactions at the osteocalcin VDRE. Furthermore, overexpression of both RXR beta and RXR alpha augmented 1,25(OH)2D3 responsiveness in transient expression studies. Direct analysis of VDRE binding in mobility shift assays demonstrated that heteromeric interactions between VDR and RXR were enhanced by 1,25(OH)2D3 and were not affected appreciably by 9-cis retinoic acid, except that inhibition was observed at high retinoid concentrations. These data suggest a regulatory mechanism for osteocalcin gene expression that involves 1,25(OH)2D3-induced heterodimerization of VDR and unliganded RXR. 9-cis retinoic acid may attenuate 1,25(OH)2D3 responsiveness by diverting RXRs away from VDR-mediated transcription and towards other RXR-dependent transcriptional pathways.

cAMP-dependent plasticity at excitatory cholinergic synapses in Drosophila neurons: alterations in the memory mutant dunce.

It is well known that cAMP signaling plays a role in regulating functional plasticity at central glutamatergic synapses. However, in the Drosophila CNS, where acetylcholine is thought to be a primary excitatory neurotransmitter, cellular changes in neuronal communication mediated by cAMP remain unexplored. In this study we examined the effects of elevated cAMP levels on fast excitatory cholinergic synaptic transmission in cultured embryonic Drosophila neurons. We report that chronic elevation in neuronal cAMP (in dunce neurons or wild-type neurons grown in db-cAMP) results in an increase in the frequency of cholinergic miniature EPSCs (mEPSCs). The absence of alterations in mEPSC amplitude or kinetics suggests that the locus of action is presynaptic. Furthermore, a brief exposure to db-cAMP induces two distinct changes in transmission at established cholinergic synapses in wild-type neurons: a short-term increase in the frequency of spontaneous action potential-dependent synaptic currents and a long-lasting, protein synthesis-dependent increase in the mEPSC frequency. A more persistent increase in cholinergic mEPSC frequency induced by repetitive, spaced db-cAMP exposure in wild-type neurons is absent in neurons from the memory mutant dunce. These data demonstrate that interneuronal excitatory cholinergic synapses in Drosophila, like central excitatory glutamatergic synapses in other species, are sites of cAMP-dependent plasticity. In addition, the alterations in dunce neurons suggest that cAMP-dependent plasticity at cholinergic synapses could mediate changes in neuronal communication that contribute to memory formation.

Biological consequences of a reduction in the non-target DNA scanning capacity of a DNA repair enzyme.

Numerous DNA-interactive proteins have been shown to locate specific sequences within large domains of non-target DNA in vitro and in vivo by a one-dimensional diffusion mechanism; however, the biological significance of this process has not been evaluated. We have examined the biological consequences of sliding for the pyrimidine dimer-specific DNA repair enzyme T4 endonuclease V, an enzyme which scans non-target DNA both in vitro and in vivo. An endonuclease V mutant was constructed whose only altered biochemical characteristic, measured in vitro, was a loss in its ability to slide on non-target DNA. In contrast to the native enzyme, when the mutated endonuclease V was expressed in DNA repair-deficient Escherichia coli, no enhanced ultraviolet survival was conferred. These results suggest that the mechanisms which DNA-interactive proteins employ to enhance the probability of locating their target sequences are of significant biological importance.

Differential regulation and transcriptional control of immediate early gene expression in forskolin-treated WEHI7.2 thymoma cells.

Agents that increase intracellular cAMP are frequently growth inhibitory for lymphocytes and induce apoptosis in cortical thymocytes by regulating gene expression. In the present study, immediate early gene expression was examined in WEHI7.2 thymoma cells undergoing cAMP-mediated apoptosis. Temporal differences in c-fos, junB, and inducible cAMP early repressor (ICER) steady-state mRNA levels were observed after forskolin exposure. Maximal induction of c-fos and junB occurred within 1 h, returning to basal levels by 3.5 h. In contrast, a 1.5-h time lag was observed before ICER transcript levels increased, reaching maximal levels after 3.5 h. This rise in expression, correlating with the decrease in c-fos and junB levels, preceded apoptotic DNA fragmentation by 1.5 h. Transient expression of ICER promoter constructs demonstrated that cAMP responsiveness occurred through cAMP-autoregulatory response element (CARE)3/4, two of the four proposed response elements in the ICER promoter. In contrast to the cAMP-responsive cell line JEG-3, CARE1/2 was not functional for cAMP-activated transcription in WEHI7.2 cells. An observed differential binding pattern of WEHI and JEG nuclear extracts to these elements may account for the cell-specific differences in expression patterns. To determine the role of endogenous ICER in regulating gene expression, cells were treated with two sequential doses of forskolin after which ICER and c-fos mRNA levels were measured. The high levels of cAMP-induced ICER expression dramatically reduced a second induction of c-fos. These data suggest that ICER expression may function as an antioncogene to attenuate the expression of certain protooncogenes, thereby preventing transformation and oncogenesis due to continuous overexpression. Moreover, inhibition of growth-stimulatory genes may be required for the activation of the cell death machinery in specific cells.

Functional analysis of the mouse ICER (Inducible cAMP Early Repressor) promoter: evidence for a protein that blocks calcium responsiveness of the CAREs (cAMP autoregulatory elements).

Although Ca2+ and cAMP mediate their effects through distinct pathways, both signals converge upon the phosphorylation of the cAMP response element (CRE) binding protein, CREB, thereby activating transcription of CRE-regulated genes. In WEHI7.2 thymocytes, cAMP increases the expression of the inducible cAMP early repressor (ICER) gene through CRE-like elements, known as cAMP autoregulatory elements (CAREs). Because Ca2+ -and cAMP-mediated transcription converge in WEHI7.2 thymocytes, we examined the effect of Ca2+ fluxes on the expression of the ICER gene in these cells. Despite the presence of multiple CAREs within its promoter, ICER gene transcription was not activated by Ca2+. Moreover, Ca2+ attenuated the stimulatory effect of cAMP on ICER expression. Transient expression of reporter constructs demonstrated that when these CAREs were placed in a different DNA promoter context, the elements became responsive to Ca2+. Detailed studies using chimeric promoter constructs to map the region responsible for blocking the transcriptional response to Ca2+ indicated that a small portion of the ICER promoter was necessary for the effect. Southwestern blot analysis identified a 83-kDa nuclear protein that bound specifically to that region. The relative binding activity of the factor to the ICER promoter and mutant promoter sequences correlated with an inhibition of Ca2+ -activated gene expression in WEHI7.2 cells. These data suggest that the factor functions as a putative Ca2+ -activated repressor of CREB/CRE-mediated transcription. Thus, depending on the surrounding context in which the CRE is located, CREs of individual genes can be regulated separately by Ca2+ and cAMP despite the convergence of these two signaling pathways.

Stable expression of the calbindin-D28K complementary DNA interferes with the apoptotic pathway in lymphocytes.

The WEHI7.2 thymoma cell line undergoes apoptotic cell death when exposed to glucocorticoids and agents that increase intracellular cAMP. Several lines of evidence indicate that calcium may play an important role in events culminating in lymphocyte apoptosis. In these studies, calbindin-D28K was stably overexpressed in WEHI7.2 cells to determine if increasing the Ca(2+)-binding capacity of the cell interferes with the apoptotic pathway. Indeed, stable expression of calbindin-D28K decreased the apoptotic effects of dexamethasone and forskolin, and the level of resistance to these agents correlated with the relative amount of calbindin expressed in each line. Overexpression of calbindin also increased cell survival in the presence of the calcium ionophore A23187. The stably expressed calcium-binding protein appeared to exert its protective effect subsequent to transcriptional activation, since glucocorticoid- and cAMP-induced gene expression were not affected. These data support the proposal that calcium fluxes are involved in apoptosis and suggest that high level expression of proteins that buffer calcium fluxes can effectively suppress death in apoptosis-susceptible cells.

Single-cell analysis of gene expression in the nervous system. Measurements at the edge of chaos.

The characteristic functions of tissues and organs result from the integrated activity of individual cells. Nowhere is this more evident than in the nervous system, where the activities of single neurons communicating via electrical and chemical signals mediate complex functions, such as learning and memory. The past decade has seen an explosion in the identification of genes encoding proteins, such as voltage-gated channels and neurotransmitter receptors, responsible for neuronal excitability. These studies have highlighted the fact that even within a neuroanatomically defined region, the coexistence of multiple cell types makes it difficult, if not impossible, to correlate patterns of gene expression with function. The recent development of techniques sensitive enough to study gene expression at the single-cell level promises to break this bottleneck to our further understanding. Using examples taken from our own laboratories and the work of others, we review these techniques, their application, and discuss some of the difficulties associated with the interpretation of the data.

Regulation of alpha7 nicotinic acetylcholine receptors in mouse somatosensory cortex following whisker removal at birth.

Previous studies in postnatal mouse demonstrating high levels of alpha7 nicotinic acetylcholine receptors on layer IV somatosensory cortical neurons coincident with the onset of functional synaptic transmission led us to investigate whether the number and/or the localization of these receptors could be regulated by activity. Accordingly, we examined alpha-bungarotoxin binding in mouse somatosensory cortex following removal of all of the vibrissae on one side of the face, either by vibrissal follicle cauterization or daily plucking beginning on the day of birth. Following vibrissa plucking, the levels of [125I]alpha-bungarotoxin binding on postnatal day 6 were significantly higher (23 +/- 7%) in the denervated cortex (contralateral to the peripheral manipulation) than the intact cortex. Cauterization also resulted in significantly higher (14 +/- 3%) [125I]alpha-bungarotoxin binding in the contralateral vs. the ipsilateral cortex. In contrast, there was no difference in [125I]alpha-bungarotoxin binding in the left and right cortices of unoperated control animals. At postnatal day 14, levels of [125I]alpha-bungarotoxin binding in layer IV were very low in control animals as well as in animals subjected to whisker plucking or cautery. These findings suggest that reducing activity in the somatosensory pathway regulates the density of alpha7 nicotinic acetylcholine receptors during the first postnatal week. However, the normal decrease in receptor density that is seen during the second postnatal week of development proceeds despite altered sensory activity.

Localization of alpha 7 nicotinic receptor subunit mRNA and alpha-bungarotoxin binding sites in developing mouse somatosensory thalamocortical system.

Previous studies in rat, showing a transient pattern of expression of the alpha 7 nicotinic acetylcholine receptor in the ventrobasal thalamus and barrel cortex during the first 2 postnatal weeks, suggest that these receptors may play a role in development of the thalamocortical system. In the present study, in situ hybridization and radiolabeled ligand binding were employed to examine the spatiotemporal distribution of alpha 7 mRNA and alpha-bungarotoxin binding sites in the thalamocortical pathway of mouse during early postnatal development. As in the rat, high levels of alpha 7 mRNA and alpha-bungarotoxin binding sites are present in the barrel cortex of mouse during the first postnatal week. Both alpha 7 mRNA and its receptor protein are observed in all cortical laminae, with the highest levels seen in the compact cortical plate, layer IV, and layer VI. When viewed in a tangential plane, alpha 7 mRNA and alpha-bungarotoxin binding sites delineate a whisker-related barrel pattern in layer IV by P3-5. Quantitative analysis reveals a dramatic decrease in the levels of expression of alpha 7 mRNA and alpha-bungarotoxin binding sites in the cortex by the end of the second postnatal week. Unlike in the rat, only low levels of alpha 7 mRNA or alpha-bungarotoxin binding sites are present in the ventrobasal complex of the mouse thalamus. The broad similarities between the thalamocortical development of rat and mouse taken together with the present results suggest that alpha 7 receptors located on cortical neurons, rather than on thalamic neurons, play a role in mediating aspects of thalamocortical development.

Immunofluorescent evidence for exocytosis and internalization of secretory granule membrane in isolated chromaffin cells.

Cultured bovine adrenal medullary chromaffin cells were stimulated with the secretogogues Ba2+ or carbamyl choline plus Ca2+ in the presence of a monospecific rabbit IgG fraction directed against bovine dopamine beta-hydroxylase. The anti-dopamine beta-hydroxylase was labeled either with fluorescent protein A or with a fluorescent second antibody to rabbit IgG. Stimulation produced a patchy cell surface distribution of fluorescence. There was no noticeable internalization of the fluorescence for up to 2 h. In similar experiments using fluorescent monovalent fragments (Fab) of the same monospecificidopamine-beta-hydroxylase IgG, a more uniform distribution of the fluorescence was observed. A few min after a 5 min period of stimulation with Ba2+, the fluorescence appeared to be on or near the cell surface; however, after 20 min or more it was distributed throughout the cytoplasm except that the cell nuclei were not labeled. Thus, dopamine beta-hydroxylase which appeared on the cell surface as a consequence of exocytosis was internalized in the presence of monovalent antibody fragments, but not in the presence of the divalent (polyclonal) antibody, presumably because endocytosis of dopamine beta-hydroxylase was inhibited by crosslinking of the dopamine beta-hydroxylase molecules. The internalized anti-dopamine beta-hydroxylase Fab fragments were found to reappear on the cell surface during a second secretory response. It is concluded that the interior of the chromaffin granule membrane, for which dopamine beta-hydroxylase is a marker, becomes exposed on the surface of the cell during secretion and that the membrane is then retrieved back into the cell where it can be re-used in a further secretory cycle.

Agrin gene expression in mouse somatosensory cortical neurons during development in vivo and in cell culture.

Agrin is an extracellular matrix protein involved in the formation of the postsynaptic apparatus of the neuromuscular junction. In addition to spinal motor neurons, agrin is expressed by many other neuronal populations throughout the nervous system. Agrin's role outside of the neuromuscular junction, however, is poorly understood. Here we use the polymerase chain reaction to examine expression and alternative splicing of agrin in mouse somatosensory cortex during early postnatal development in vivo and in dissociated cell culture. Peak levels of agrin gene expression in developing cortex coincide with ingrowth of thalamic afferent fibres and formation of thalamocortical and intracortical synapses. Analysis of alternatively spliced agrin messenger RNA variants shows that greater than 95% of all agrin in developing and adult somatosensory cortex originates in neurons, including isoforms that have little or no activity in acetylcholine receptor aggregation assays. The levels of expression of "active" and "inactive" isoforms, however, are regulated during development. A similar pattern of agrin gene expression is also observed during a period when new synapses are being formed between somatosensory neurons growing in dissociated cell culture. Changes in agrin gene expression, observed both in vivo and in vitro, are consistent with a role for agrin in synapse formation in the central nervous system.

Crosstalk during Ca2+-, cAMP-, and glucocorticoid-induced gene expression in lymphocytes.

In the WEHI7.2 thymoma cell line, cAMP, glucocorticoids, or increases in cytosolic Ca2+ concentration lead to cell death by apoptosis. In the present study, we examined the effects of these compounds on cAMP response element (CRE)-mediated gene expression. Thapsigargin and A23187 were employed to increase cytosolic Ca2+ levels and induce apoptosis. Both compounds enhanced transcription from a CRE preceding apoptotic death. Moreover, the transcriptional response to the combination of forskolin and either thapsigargin or A23187 was synergistic mirroring the effect on cell death. Importantly, dexamethasone treatment, which causes an efflux of Ca2+ from the ER, induced transcription from a CRE alone or in synergy with forskolin. The increase in CRE-controlled gene expression correlated with a decrease in cell viability. Following treatment with forskolin, thapsigargin, or dexamethasone, the CRE binding protein (CREB) was phosphorylated at levels correlating with the level of induced gene expression. These data suggest that transcriptional crosstalk between independent signaling pathways occurs in lymphocytes, and CREB may play a central role in the mediation of CRE-dependent transcription by these diverse set of apoptotic agents.