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We previously reported 12 antioxidative flavonoids isolated from the n-BuOH extract of Inula britannica (Asteraceae). This prompted us to investigate further whether these flavonoids also showed antioxidative activity upon live cells grown in a culture system. Among the 12 flavonoids tested, only patuletin, nepetin, and axillarin protected primary cultures of rat cortical cells from oxidative stress induced by glutamate. These flavonoids exerted significant neuroprotective activity when they were administered either before or after the glutamate insult. Treatment with these flavonoids maintained the activities of such antioxidant enzymes as catalase, glutathione-peroxidase, and glutathione reductase, all of which play important roles in the antioxidative defense mechanism. Moreover, these three flavonoids also attenuated significant drops in glutathione induced by glutamate which is a routine concomitant of oxidative stress by inhibiting glutathione diminution. Accordingly, these flavonoids did not stimulate the synthesis of glutathione. With regard to structure-activity relationships, our results indicated that the 6-methoxyl group in the A ring and the 3', 4'-hydroxyl groups in the B ring are crucial for the protection against the oxidative stress; glycosylation greatly reduced their protective activities. Collectively, these results indicated that patuletin, nepetin, and axillarin strongly protect primary cultured neurons against glutamate-induced oxidative stress.

There is increasing evidence that estrogen influences electrical activity of neurons via stimulation of membrane receptors. Although the presence of intracellular estrogen receptors and their responsiveness in dorsal root ganglion (DRG) primary sensory neurons were reported, rapid electrical responses of estrogen in DRG neurons have not been reported yet. Therefore the current study was initiated to examine the rapid effects of estrogen on Ca2+ channels and to determine its detailed mechanism in female rat DRG neurons using whole-cell patch-clamp recordings. Application of 17beta-estradiol (1 microM) caused a rapid inhibition on high-voltage-activated (HVA)-, but not on low-voltage-activated (LVA)-Ca2+ currents. This rapid estrogen-mediated inhibition was reproducible and dose-dependent. This effect was also sex- and stereo-specific; it was greater in cells isolated from intact female rats and was more effective than that of 17alpha-estradiol, the stereoisomer of the endogenous 17alpha-estradiol. In addition, ovariectomy reduced the inhibition significantly but this effect was restored by administration of estrogen in ovariectomized subjects. Occlusion experiments using selective blockers revealed 17beta-estradiol mainly targeted on both L- and N-type Ca2+ currents. Overnight treatment of cells with pertussis toxin profoundly reduced 17beta-estradiol-mediated inhibition of the currents. On the other hand, estradiol conjugated to bovine serum albumin (EST-BSA) produced a similar extent of inhibition as 17beta-estradiol did. These results suggest that 17beta-estradiol can modulate L- and N-type HVA Ca2+ channels in rat DRG neurons via activation of pertussis toxin-sensitive G-protein(s) and non-genomic pathways. It is likely that such effects are important in estrogen-mediated modulation of sensory functions at peripheral level.

1. We previously reported that four new phenylpropanoid glycosides and six known cinnamate derivatives isolated from roots of Scrophularia buergeriana Miquel (Scrophulariaceae) protected cultured cortical neurons from neurotoxicity induced by glutamate. Here, we have investigated the structure-activity relationships in the phenylpropanoids using our primary culture system. 2. The alpha,beta-unsaturated ester moiety and the para-methoxy group in the phenylpropanoids appeared to play a vital role in neuroprotective activity. This suggested that E-p-methoxycinnamic acid (E-p-MCA) might be a crucial component for their neuroprotective activity within the phenylpropanoid compounds. E-p-MCA significantly attenuated glutamate-induced neurotoxicity when added prior to an excitotoxic glutamate challenge. 3. The neuroprotective activity of E-p-MCA appeared to be more effective in protecting neurons against neurotoxicity induced by NMDA than from that induced by kainic acid. E-p-MCA inhibited the binding of [propyl-2,3-(3)H]-CGP39653 and [2-(3)H]-glycine to their respective binding sites on rat cortical membranes. However, even high concentrations of E-p-MCA failed to inhibit completely [propyl-2,3-(3)H]-CGP39653 and [2-(3)H]-glycine binding. 4. Indeed, E-p-MCA diminished the calcium influx that routinely accompanies glutamate-induced neurotoxicity, and inhibited the subsequent overproduction of nitric oxide and cellular peroxide in glutamate-injured neurons. 5. Thus, our results suggest that E-p-MCA exerts significant protective effects against neurodegeneration induced by glutamate in primary cultures of cortical neurons by an action suggestive of partial glutamatergic antagonism.

There is increasing evidence that ginseng influences pain modulation. In spite of extensive behavior studies, the detailed mechanism of ginseng actions at the cellular level and the identity of the active substance have not been elucidated yet. Whole-cell patch-clamp recordings were used to examine the modulation of high-voltage-activated Ca2+ channel currents by ginseng total saponins and its various individual ginsenosides in rat dorsal root ganglion neurons. Application of ginseng total saponins suppressed Ca2+ channel currents in a dose-dependent manner. Occlusion experiments using selective blockers revealed that ginseng total saponins could modulate L-, N-, and P-type currents. The co-application of ginseng total saponins and the gamma-opioid receptor agonist, D-Ala(2), N-MePhe(4), Gly(5)-ol-enkephalin (DAMGO), produced non-additive effects in most cells tested and each effect was significantly relieved by a depolarizing prepulse. Overnight treatment of cells with pertussis toxin profoundly reduced the inhibition. Furthermore, we now report that ginsenoside Rg3, among the major fractions of ginseng saponins, is a newly identified active component for the inhibition. These results suggest that the modulation of Ca2+ channels by ginseng total saponins, in particular by ginsenoside Rg3, could be part of the pharmacological basis of ginseng-mediated antinociception.

Etoposide-induced death comprises such nuclear events as the formation of topoisomerase II-DNA cleavable complex and cytosolic events including caspase activation. By first establishing the temporospatial death sequence triggered by etoposide in a neuronal cell line, MN9D overexpressing Bcl-X(L) (MN9D/Bcl-X(L)) or control vector (MN9D/Neo), we examined whether formation of this complex is primarily responsible for cell death and at which strategic points and how Bcl-X(L) blocks etoposide-induced neuronal death. Etoposide induced death that was dependent on caspase, cycloheximide, and calpain in MN9D/Neo cells. Etoposide also induced death in enucleated MN9D/Neo cells, although this was less severe. The level of topoisomerase II-DNA cleavable complex reached at a maximum of 2 hr after etoposide treatment was identical in MN9D/Neo and MN9D/Bcl-X(L) cells. In MN9D/Neo cells, cytochrome c release into the cytosol and caspase activation occurred as early as 2 hr and 3-6 hr after etoposide treatment, respectively. Etoposide-induced DNA laddering potentially via caspase appeared as early as 12 hr after drug treatment, followed by nuclear swelling in MN9D/Neo cells (>18-20 hr). Subsequently, nuclear condensation started by 24-28 hr and became apparent thereafter. All of these events except for nuclear swelling were substantially blocked in MN9D/Bcl-X(L). At the later stage of cell death (<32-36 hr), a specific cleavage of Bax and fodrin appeared that was completely blocked by calpain inhibitor or by Bcl-X(L). Taken together, our data suggest that Bcl-X(L) prevents etoposide-induced neuronal death by exerting its anticaspase and anticalpain effect on cellular events after the formation of topoisomerase II-DNA cleavable complex that may not be a major contributor to cell death., (Copyright 2001 Wiley-Liss, Inc.)

An MN9D dopaminergic neuronal cell line overexpressing calbindin-D28K (MN9D/Calbindin) was established in order to investigate directly the potential role of calcium-binding protein in neuronal differentiation. Overexpression of calbindin-D28K in MN9D cells resulted in significant increases in the number of neurites, the length of primary neurites, and the total extent of neurites. This robust neurite outgrowth occurred without cessation of cell division. Analysis of immunoblots revealed that this morphological differentiation was accompanied by increased expression of such markers of maturation as the synaptosomal protein SNAP-25. During calbindin-D28K-evoked neurite outgrowth in MN9D cells, phosphorylation of p38 mitogen-activated protein kinase (MAPK) dramatically increased while the levels and extent of phosphorylation of such other MAPKs as c-Jun N-terminal kinase (JNK) or extracellular response kinase (ERK) were not altered. Consequently, calbindin-D28K-induced neurite outgrowth was largely abolished by treatment with a p38 inhibitor, PD 169316, while the level of SNAP-25 in MN9D/Calbindin cells was not altered by this treatment. These data support an idea that calbindin-D28K and its associated p38 signaling pathway play a role in dopaminergic neuronal differentiation., (Copyright 2001 Academic Press.)

The effects of neurotoxins on levels of mitochondrially encoded gene transcripts in a dopaminergic neuronal cell line, MN9D, were examined following treatment with 200 microM N-methyl-4-phenylpyridinium (MPP(+)) or 6-hydroxydopamine (6-OHDA). As confirmed by a Northern blot analysis, levels of cytochrome c oxidase subunit 3 (COX III) and ATPase subunit 6 (ATPase 6) transcript were decreased in a time-dependent manner following treatment with MPP(+) but not with 6-OHDA. Accordingly, enzymatic activity of cytochrome c oxidase (COX) and the intracellular ATP content were also decreased in MPP(+)-treated cells while these remained unaltered in 6-OHDA-treated cells. In the cell death paradigm induced by MPP(+), overexpression of Bcl-2 in MN9D cells (MN9D/Bcl-2) significantly blocked MPP(+)-induced downregulation of COX III and ATPase 6 transcripts. In MN9D/Bcl-2 cells, MPP(+)-induced downregulation of COX activity and the intracellular level of ATP was also blocked. Treatment with a pan-caspase inhibitor, however, neither prevented MPP(+)-induced downregulation of COX activity nor affected intracellular level of ATP in MN9D cells. Taken together, our present data suggest that Bcl-2 may play a regulatory role in energy metabolism by preventing downregulation of mitochondrially encoded gene(s) at a point distinct from its known anticaspase activity in MPP(+)-induced dopaminergic neuronal death., (Copyright 2001 Academic Press.)

Two cysteine protease families, caspase and calpain, are known to participate in cell death. We investigated whether a stress-specific protease activation pathway exists, and to what extent Bcl-2 plays a role in preventing drug-induced protease activity and cell death in a dopaminergic neuronal cell line, MN9D. Staurosporine (STS) induced caspase-dependent apoptosis while a dopaminergic neurotoxin, MPP(+) largely induced caspase-independent necrotic cell death as determined by morphological and biochemical criteria including cytochrome c release and fluorogenic caspase cleavage assay. At the late stage of both STS- and MPP(+)-induced cell death, Bax was cleaved into an 18-kDa fragment. This 18-kDa fragment appeared only in the mitochondria-enriched heavy membrane fraction of STS-treated cells, whereas it was detected exclusively in the cytosolic fraction of MPP(+)-treated cells. This proteolytic cleavage of Bax appeared to be mediated by calpain as determined by incubation with [(35)S]methionine-labelled Bax. Thus, cotreatment of cells with calpain inhibitor blocked both MPP(+)- and STS-induced Bax cleavage. Intriguingly, overexpression of baculovirus-derived inhibiting protein of caspase, p35 or cotreatment of cells with caspase inhibitor blocked STS- but not MPP(+)-induced Bax cleavage. This appears to indicate that calpain activation may be either dependent or independent of caspase activation within the same cells. However, cotreatment with calpain inhibitor rescued cells from MPP(+)-induced but not from STS-induced neuronal cell death. In these paradigms of dopaminergic cell death, overexpression of Bcl-2 prevented both STS- and MPP(+)-induced cell death and its associated cleavage of Bax. Thus, our results suggest that Bcl-2 may play a protective role by primarily blocking drug-induced caspase or calpain activity in dopaminergic neuronal cells.

Regulation of intracellular calcium levels plays a central role in cell survival, proliferation, and differentiation. A cell-permeable, tumor-promoting thapsigargin elevates the intracellular calcium levels by inhibiting endoplasmic reticulum Ca(2+)-ATPase. The Src-tyrosine kinase family is involved in a broad range of cellular responses ranging from cell growth and cytoskeletal rearrangement to differentiation. The immediate early gene pip92 is induced in neuronal cell death as well as cell growth and differentiation. To resolve the molecular mechanism of cell growth by intracellular calcium mobilization, we have examined the effect of thapsigargin and subsequent intracellular calcium influx on pip92 expression in immortalized rat hippocampal H19-7 cells. An increase of intracellular calcium ion levels induced by thapsigargin stimulated the expression of pip92 in H19-7 cells. Transient transfection of the cells with kinase-inactive mitogen-activated protein kinase kinase (MEK) and Src kinase or pretreatment with the chemical MEK inhibitor PD98059 significantly inhibited pip92 expression induced by thapsigargin. When constitutively active v-Src or MEK was overexpressed, the transcriptional activity of the pip92 gene was markedly increased. Dominant inhibitory Raf-1 blocked the transcriptional activity of pip92 induced by thapsigargin. The transcription factor Elk1 is activated during thapsigargin-induced pip92 expression. Taken together, these results suggest that an increase of intracellular calcium ion levels by thapsigargin stimulates the pip92 expression via Raf-MEK-extracellular signal-regulated protein kinase- as well as Src kinase-dependent signaling pathways.

We investigated the role of tumor necrosis factor (TNF)-alpha in the onset of neuronal and glial apoptosis after traumatic spinal cord crush injury in rats. A few TUNEL-positive cells were first observed within and surrounding the lesion area 4 h after injury, with the largest number observed 24-48 h after injury. Double-labeling of cells using cell type-specific markers revealed that TUNEL-positive cells were either neurons or oligodendrocytes. One hour after injury, an intense immunoreactivity to TNF-alpha was observed in neurons and glial cells in the lesion area, but also seen in cells several mm from the lesion site rostrally and caudally. The level of nitric oxide (NO) also significantly increased in the spinal cord 4 h after injury. The injection of a neutralizing antibody against TNF-alpha into the lesion site several min after injury significantly reduced both the level of NO observed 4 h thereafter as well as the number of apoptotic cells observed 24 h after spinal cord trauma. An inhibitor of nitric oxide synthase (NOS), N(G)-monomethyl-l-arginine acetate (l-NMMA), also reduced the number of apoptotic cells. This reduction of apoptotic cells was associated with a decrease in DNA laddering on agarose gel electrophoresis. These results suggest that: (i) TNF-alpha may function as an external signal initiating apoptosis in neurons and oligodendrocytes after spinal cord injury; and (ii) TNF-alpha-initiated apoptosis may be mediated in part by NO as produced by a NOS expressed in response to TNF-alpha., (Copyright 2000 Academic Press.)

In higher vertebrates, reactive gliosis resulting from injury to the central nervous system (CNS) is characterized by a rapid increase in immunoreactivity (IR) to glial fibrillary acidic protein (GFAP). Little is known about the extracellular signals that initiate the increase in GFAP-IR following CNS injury. We demonstrated recently [T.H. Oh, G.J. Markelonis, J.R. Von Visger, B. Baik, M.T. Shipley, Acidic pH rapidly increases immunoreactivity of glial fibrillary acidic protein in cultured astrocytes, Glia 13 (1995) 319-322] that a rapid increase in GFAP-IR can be evoked in mature astrocyte cultures by exposing the cells to an acidic medium. We investigated the intracellular pathway(s) involved in initiating increased GFAP-IR, a hallmark of reactive astrocytes. The increase in GFAP-IR produced by exposure to acidic medium was blocked by pretreatment with nickel ions, by such blockers of L-type calcium channels as nifedipine, verapamil and diltiazem, by calpain inhibitor I, or by the intracellular calcium chelator, BAPTA-AM. At physiological pH, treatment with the calcium ionophore, A23187, resulted in increased GFAP-IR which could be blocked by pretreatment with calpain inhibitor I. Astrocytes exposed to low pH exhibited a marked increase in a GFAP fragment with a molecular weight of 48 kDa. In astrocytes exposed to acidic medium, alpha-fodrin, a selective endogenous substrate of calpain, was also found to be hydrolyzed producing fragments with molecular weights of 120-150 kDa. As anticipated, pretreatment with calpain inhibitor I prevented the proteolytic degradation of both GFAP and alpha-fodrin in these samples. These results suggest that the initial increase in GFAP-IR after CNS injury appears to be linked to Ca(++) influx, and is mediated further by a proteolytic process that seemingly involves the activation of the calcium-dependent protease, calpain I.

A rapid and sensitive indirect competitive enzyme immunoassay method has been developed for quantitating ginsenoside Rf (Rf) in crude total Panax ginseng saponins and in rat plasma using high titer mouse monoclonal antibody (mAb) raised against a conjugate of Rf and bovine serum albumin (BSA). The isotype of mAb against Rf was IgG3 with a K chain. The presence of Rf inhibited the binding of the mouse anti-Rf mAb to a Rf-BSA solid phase coating antigen. The working range was 0.01-10 ng/assay and detection limits were 20 pg in various ginseng extract fractions or 34 pg in rat plasma per assay. The anti-Rf mAb cross-reacted with ginsenoside Rg2 by 57.5%, but not with other ginsenosides. However, this anti-Rf mAb did not cross-react with BSA or cellubiose, which is a carbohydrate component of Rf. Using this standard curve, we could measure the amount of Rf in ginseng total extract, ginseng total saponins, protopanaxadiol saponins, and propanaxatriol saponins. We could also measure the amount of Rf in rat plasma after the oral administration of Rf and found that Rf reached a maximum level in rat plasma after 16 h. These results indicate that the anti-Rf mAb could be useful for the quantitation of Rf in crude ginseng fractions and in body fluids.

To examine the correlation between the structure of Bcl-2 and its inhibitory function of c-Jun N-terminal kinase (JNK) and caspase activity, we established a dopaminergic neuronal cell line, MN9D overexpressing Bcl-2 (MN9D/Bcl-2) or its structural mutants. The mutants comprised a point mutation in the BH1 (G145A; MN9D/BH1) or BH2 (W188A; MN9D/BH2) domain and a deletion mutation in the C-terminal (MN9D/C22), BH3 (MN9D/BH3), or BH4 (MN9D/BH4) domain. As determined by the TUNEL (terminal deoxynucleotidyltransferase nick end-labeling) and MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] reduction assay, apoptotic death of MN9D/Neo cells reached 80-90% within 24 h in response to 1 microM staurosporine. Upon staurosporine treatment, JNK activity increased six- to sevenfold over the basal level within 2-4 h. Treatment of MN9D/Neo with both staurosporine and a caspase inhibitor, Z-VAD, attenuated cell death without suppressing JNK activation. Both staurosporine-induced cell death and JNK activation were attenuated in MN9D/Bcl-2. As determined by cleavage of poly(ADP-ribose) polymerase into 85 kDa, Bcl-2 blocked caspase activity as well. When cells overexpressing one of the Bcl-2 mutants were treated with staurosporine, death was attenuated in MN9D/BH1, MN9D/BH2, and MN9D/C22 but not in MN9D/BH3 and MN9D/BH4. Similarly, both JNK and caspase activation were blocked in MN9D/BH1, MN9D/BH2, and MN9D/C22, whereas they were not suppressed in MN9D/BH3 and MN9D/BH4. Taken together, our data indicate that there exists a close structural and functional correlation of Bcl-2 to JNK and caspase activity in staurosporine-induced dopaminergic neuronal cell death.

This self-directed learning module highlights assessment and therapeutic options in the rehabilitation of patients with osteoarthritis. It is part of the chapter on rehabilitation of orthopedic and rheumatologic disorders in the Self-Directed Physiatric Education Program for practitioners and trainees in physical medicine and rehabilitation. New advances covered in this article include updates on conservative and operative treatment of lumbar spinal stenosis and pediatric hip diseases, prophylactic therapy for thromboembolic disease after lower limb joint replacement, new therapies for osteoarthritis, and the impact of exercise on outcome following hip replacement in active persons.

This self-directed learning module highlights assessment and therapeutic options in the rehabilitation of patients with connective tissue diseases. It is part of the chapter on rehabilitation of orthopedic and rheumatologic disorders in the Self-Directed Physiatric Education Program for practitioners and trainees in physical medicine and rehabilitation. New advances covered in this article include the management of patients who have polymyalgia rheumatica, juvenile rheumatoid arthritis, ankylosing spondylitis, rheumatoid cervical spine, and rheumatoid hand.

This self-directed learning module highlights assessment and therapeutic options in the rehabilitation of patients with orthopedic and musculoskeletal disorders. It is part of the chapter on rehabilitation of orthopedic and rheumatologic disorders in the Self-Directed Physiatric Education Program for practitioners and trainees in physical medicine and rehabilitation. This article discusses new advances in such topics as idiopathic scoliosis, nontraumatic shoulder pain, rotator cuff tendinitis, and Dupuytren's disease.

This self-directed learning module reviews and summarizes recent literature on osteoporosis. It is part of the chapter on rehabilitation of orthopedic and rheumatologic disorders in the Self-Directed Physiatric Education Program for practitioners and trainees in physical medicine and rehabilitation. The areas covered include pathophysiology of primary and secondary osteoporosis, effects of various pharmacologic treatments on bone metabolism, and the utility of available diagnostic tests. Management strategies for perimenopausal women as compared with postmenopausal women with established osteoporosis are discussed. This is followed by an evaluation and management plan for the older man with acute osteoporotic fracture.

Oxidative stress has been implicated as a primary cause of neuronal death in certain neurodegenerative disorders and in aging brains. Natural products have been used in Asian societies for centuries for treating such neurodegenerative disorders as senile dementia. In an effort to identify active neuroprotective compounds from these products, we have employed cultures of rat cortical neurons as our screening system. A methanolic extract from dried roots of Cynanchum wilfordii Hemsley (Asclepiadaceae) significantly mitigated the neurotoxicity induced by H2O2 in this screening system. Activity-guided fractionation using several chromatographic techniques resulted in the isolation of the neuroprotective compound, cynandione A, a biacetophenone. At a concentration of 50 microM, cynandione A significantly reduced neurotoxicity induced by H2O2. Cynandione A significantly attenuated decreases in levels of glutathione, superoxide dismutase, and other enzymes that participate in the cellular defense against oxidative stress. Furthermore, cynandione A alleviated neurotoxicity induced by the excitotoxic neurotransmitter, L-glutamate, the neurotoxicity induced by kainate, but not that mediated by N-methyl-D-aspartate. Cynandione A was demonstrated to be a natural antioxidant as it facilitated the breakdown of hydrogen peroxide in vitro; however, no mechanism was uncovered to explain its neuroprotectant effects against glutamate and kainate. Therefore, cynandione A may be efficacious in protecting neurons from oxidative stress mediated via activation of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate/kainate receptors since it exerted significant neuroprotective effects on cultured cortical neurons.

To further characterize MPP(+)-induced cell death and to explore the role of Bcl-2-related proteins in this death paradigm, we utilized a mesencephalon-derived dopaminergic neuronal cell line (MN9D) stably transfected with human bcl-2 (MN9D/Bcl-2), its C-terminal deletion mutant (MN9D/Bcl-2Delta22), murine bax (MN9D/Bax), or a control vector (MN9D/Neo). As determined by electron microscopy and TUNEL assay, MN9D/Neo cells exposed to MPP(+) underwent a cell death that was characterized by mitochondrial swelling and irregularly scattered heterochromatin without accompanying DNA fragmentation. However, cell swelling typically seen in necrosis did not appear. To examine the biochemical events associated with MPP(+)-induced cell death, various analyses were conducted. Addition of a broad-spectrum caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (50-400 microM) or Boc-aspartyl(OMe)-fluoromethylketone (50-200 microM) did not attenuate MPP(+)-induced cell death while the same treatment protected MN9D/Neo cells against staurosporine-induced apoptotic cell death. Concurrent treatment with an inhibitor of macromolecule synthesis such as cycloheximide, emetine, or actinomycin D blocked MPP(+)-induced cell death, suggesting that new protein synthesis is required as demonstrated in many apoptotic cell death. The level of cytosolic calcium in MN9D/Neo cells was unchanged over 24 h following MPP(+) treatment, as monitored by means of the fluorescent probe Fura-2. Western blot analysis indicated that expression level of proapoptotic protein, Bax was not significantly altered after MPP(+) treatment. In this death paradigm, overexpression of Bcl-2 but not its C-terminal deletion mutant attenuated MPP(+)-induced cell death whereas overexpression of Bax had no effect. Taken together, these data indicate that (i) MPP(+) induces a distinct form of cell death which resembles both apoptosis and necrosis; and (ii) full-length Bcl-2 counters MPP(+)-induced morphological changes and cell death via a mechanism that is dependent on de novo protein synthesis but independent of cytosolic calcium changes, Bax expression, and/or activation of caspase(s) in MN9D cells., (Copyright 1999 Academic Press.)

This study was conducted to evaluate the antibody levels of alpha-toxin, capsular polysaccharides (CPS) and fibronectin-binding protein (FnBP) in rabbits immunized with an experimental vaccine against Staphylococcus aureus and to develop the bovine mastitis subunit vaccine in the future. Enzyme immunoassay was used for detection of IgG antibodies against staphylococcal CPS, alpha-toxin and FnBP. The levels of specific antibodies against CPS, alpha-toxin and FnBP in immunized rabbits were significantly increased after first immunization compared with control animals (p<0.05). Of three antigen used in vaccine, immunogenicity of CPS was relatively lower, compared with those of alpha toxin and fibronectin binding protein. Numbers of S. aureus in blood of immunized groups were lower than those of control group after bacterial challenge. But the bacterial numbers among immunized groups were not significantly different. S. aureus counts in excised organs were significantly lower in all immunized rabbits than in PBS-control group (p<0.05). The present study showed that alpha-toxin, capsular polysaccharide and fibronectin binding protein included in a subunit vaccine were protective.

In this study, we examined the possibility that MPTP and 6-hydroxydopamine (6-OHDA) act on distinct cell death pathways in a murine dopaminergic neuronal cell line, MN9D. First, we found that cells treated with 6-OHDA accompanied ultrastructural changes typical of apoptosis, whereas MPP+ treatment induced necrotic manifestations. Proteolytic cleavage of poly-(ADP-ribose)polymerase by caspase was induced by 6-OHDA, whereas it remained uncleaved up to 32 h after MPP+ treatment and subsequently disappeared. Accordingly, 6-OHDA- but not MPP(+)-induced cell death was significantly attenuated in the presence of a broad-spectrum caspase inhibitor, N-benzyloxy-carbonyl-Val-Ala-Asp-fluomethylketone (Z-VAD-fmk). As measured by fluorometric probes, the level of reactive oxygen species (ROS) significantly increased after 6-OHDA treatment. In contrast, the level of dihydroethidium-sensitive ROS following MPP+ treatment remained unchanged while a slight increase in dichlorofluorescin-sentive ROS was temporarily observed. As demonstrated by immunoblot analysis, the level of superoxide dismutase was down-regulated following 6-OHDA treatment, whereas it remained unchanged after MPP+ treatment. Cotreatment of cells with antioxidants such as N-acetylcysteine or Mn(III)tetrakis(4-benzoic acid)porphyrin chloride (MnTBAP, cell-permeable superoxide dismutase mimetic) rescued 6-OHDA- but not MPP(+)-induced cell death, whereas inclusion of catalase or N(G)-nitro-L-arginine had no effect in both cases. In addition, 6-OHDA induced ROS-mediated c-Jun N-terminal kinase (JNK) activation that was attenuated in the presence of N-acetylcysteine or MnTBAP but not catalase or Z-VAD-fmk. In contrast, MPP+ has little effect on JNK activity, indicating that ROS and/or ROS-induced cell death signaling pathway seems to play an essential role in 6-OHDA-mediated apoptosis but not in MPP(+)-induced necrosis in a mesencephalon-derived, dopaminergic neuronal cell line.

Buffering extracellular pH at the site of a spinal cord crush-injury may stimulate axonal regeneration in rats (1; Guth et al., Exp. Neurol. 88: 44-55, 1985). We demonstrated in cultured astrocytes that acidic pH initiates a rapid increase in immunoreactivity for GFAP (GFAP-IR), a hallmark of reactive gliosis (2; Oh et al., Glia 13: 319-322, 1995). We extended these studies by investigating the effects of certain treatments on reactive gliosis developing in situ in a rat spinal cord injury model. A significant reactive gliosis was observed within 2 days of cord lesion in untreated crush or vehicle-treated, crush control animals as evidenced by increased GFAP-IR and hypertrophy of astrocytes. By contrast, infusion of Pipes buffer (pH 7.4) into the lesion site significantly reduced this increase. The increased GFAP-IR appeared to be linked to Ca2+ influx since infusion of a blocker of L-type calcium channels, nifedipine, reduced the ensuing reactive gliosis significantly. While Ca2+ modulates many signaling pathways within cells, its effect on reactive gliosis appeared to result from an activation of calpain I. Calpain inhibitor I, a selective inhibitor of mu-calpain, also significantly reduced reactive gliosis. However, calpain inhibitor II, a close structural analog which blocks m-calpain, had no salutary effect. We suggest, therefore, that the initial reactive gliosis seen in vivo may result from the activation of a neutral, Ca2+-dependent protease, calpain I, through calcium influx., (Copyright 1999 Academic Press.)

While screening extracts of natural products in search of anticholinesterase activity, we found that a total methanolic extract of the tuber of Corydalis ternata (Papaveraceae) showed significant inhibitory effects on acetylcholinesterase. Further fractionation of this extract using acetylcholinesterase inhibition as the parameter screened resulted in the isolation and purification of an alkaloid, protopine. Protopine inhibited acetylcholinesterase activity in a dose-dependent manner. The concentration required for 50% inhibition was 50 microM. The anti-acetylcholinesterase activity of protopine was specific reversible and competitive in manner. Furthermore, when mice were pretreated with protopine, the alkaloid significantly alleviated scopolamine-induced memory impairment. In fact, protopine had an efficacy almost identical to that of velnacrine, a tacrine derivative developed by a major drug manufacturer to treat Alzheimer's disease, at an identical therapeutic concentration. We suggest, therefore, that protopine has both anti-acetylcholinesterase and antiamnesic properties that may ultimately hold significant therapeutic value in alleviating certain memory impairments observed in dementia.

Certain natural products and Asian herbal remedies have been used in Asia to attenuate neurodegenerative diseases, including senile dementia. We have examined derivatives of several natural products for potential neuroprotective activity in an in vitro test system. In the present study, we assayed a number of compounds that were isolated from Panax ginseng C.A. Meyer (Araliaceae) for an ability to protect rat cortical cell cultures from the deleterious effects of the neurotoxicant, glutamate. We found that ginsenosides Rb1 and Rg3 significantly attenuated glutamate-induced neurotoxicity. Brief exposure of cultures to excess glutamate caused extensive neuronal death. Glutamate-induced neuronal cell damage was reduced significantly by pretreatment with Rb1 and Rg3. Ginsenosides Rb1 and Rg3 inhibited the overproduction of nitric oxide, which routinely follows glutamate neurotoxicity, and preserved the level of superoxide dismutase in glutamate-treated cells. Furthermore, in cultures treated with glutamate, these ginsenosides inhibited the formation of malondialdehyde, a compound that is produced during lipid peroxidation, and diminished the influx of calcium. These results show that ginsenosides Rb1 and Rg3 exerted significant neuroprotective effects on cultured cortical cells. Therefore, these compounds may be efficacious in protecting neurons from oxidative damage that is produced by exposure to excess glutamate.

This self-directed learning module highlights new advances in this topic area. It is part of the chapter on rehabilitation in joint and connective tissue diseases in the Self-Directed Physiatric Education Program for practitioners and trainees in physical medicine and rehabilitation. This article discusses treatment and outcome in rheumatoid arthritis, musculoskeletal involvement in human immunodeficiency virus infection, scleroderma, systemic lupus erythematosus, and intraarticular injection of corticosteroids.

Study Objective: To elucidate risk factors for apnea in preterm infants discharged from the hospital and in full-term healthy infants. To determine the efficacy of real-time cardiopulmonary monitoring versus computerized storage and retrieval for infants at risk., Study Design: Prospective study., Setting: Operating rooms and pediatric patient care units of university medical center., Patients: 27 preterm infants and 20 full-term infants no more than 60 weeks' post-conceptional age, who were admitted for elective herniorrhaphy., Interventions: Infants were monitored before and after herniorrhaphy with general anesthesia using an infant apnea impedance monitor, pulse oximetry, and nursing observation., Measurements and Main Results: Demographic information and medical history were correlated with postoperative apnea. The sensitivity and specificity of nursing observation and oximetry were compared with computerized apnea monitors. Five patients (11%, four preterm, one full-term) were apneic postoperatively as recorded by computerized pneumocardiography. Previous apnea history, gestational age at birth, and postconceptional age at operation positively correlated with postoperative apnea. Nursing observation failed to detect 4 of 5 patients with documented apnea (sensitivity 20%, positive predictive value 50%). Pulse oximetry failed to detect 3 of 5 patients with apnea (sensitivity 40%, positive predictive value 66%)., Conclusions: Although it is easier to predict postoperative respiratory dysfunction in previously sick or very young infants, absolute predictability for all neonates remains elusive. Clinical monitors with both storage and retrieval capabilities and real-time monitoring increase our ability to detect significant events in children at risk for apnea after herniorrhaphy.

This self-directed learning module highlights new advances in this topic area. It is part of the chapter on rehabilitation in joint and connective tissue diseases in the Self-Directed Physiatric Education Program for practitioners and trainees in physical medicine and rehabilitation. This article discusses ankylosing spondylitis and spinal stenosis, including differential features, diagnostic strategy, rehabilitation management, spinal complications of ankylosing spondylitis, and pathophysiology of spinal stenosis.

This self-directed learning module highlights new advances in this topic area. It is part of the chapter on rehabilitation in joint and connective tissue diseases in the Self-Directed Physiatric Education Program for practitioners and trainees in physical medicine and rehabilitation. This article discusses the following: differential features, diagnostic strategy, and rehabilitation management of hip, knee, foot, and shoulder pain; indications, contraindications, and postsurgical management for joint arthroplasty; management of gout and chondrocalcinosis; and rehabilitation issues related to hip and shoulder fracture.

Neuroepithelial progenitor cells from forebrains of newborn rat pups develop into "mature" astrocytes in an epidermal growth factor-containing medium free of serum (Von Visger et al: Exp Neurol 128:34, 1994). Eight-week-old "mature" astrocyte cultures on poly-L-lysine-coated dishes were exposed to an acidic medium (pH 5.8-6.0) for 2-6 h. Immunoreactivity for glial fibrillary acidic protein (GFAP) dramatically and rapidly increased; this immediate increase was not affected by pretreatment with cycloheximide. In further experiments we found that the increase in GFAP was undiminished for 24-48 h after the acid-treated astrocytes were returned to normal growth medium. The Ca2+ channel antagonists nifedipine and diltiazem attenuated the increase in GFAP immunoreactivity. These results suggest that extracellular acidosis may produce a rapid increase in GFAP immunoreactivity in astrocytes independent of de novo protein synthesis, possibly by increasing intracellular levels of free Ca2+ ions.

We have studied the reactive responses of both astrocytes and microglia to dopaminergic denervation of the striatum by MPTP. Following MPTP treatment, increased GFAP immunoreactivity reached a peak at 2 days and persisted for at least 6 weeks. Immunoreactivity to vimentin was also markedly increased in astrocytes 48 h after MPTP treatment. Striatal laminin immunoreactivity, however, appeared to be unaffected by drug treatment. GFAP protein levels increased to 196% and 321% of control 24 and 48 hours after MPTP treatment, respectively. Concomitantly, GFAP mRNA levels increased to 560% and 1620% of control, respectively. These reactive changes in striatal astrocytes in response to MPTP treatment were also accompanied by a reactive microglial response as evidenced by increased immunohistochemical visualization of striatal microglia using antibodies to Mac-1. Our results and those reported previously by O'Callaghan et al., strongly suggest that MPTP-induced reactive gliosis in mouse striatum is associated with reactive microglia, albeit without increased interleukin-1 beta.

Neuroepithelial progenitor cells from striata of adult mice develop into either astrocytes or neurons when cultured in the presence of epidermal growth factor (B. A. Reynolds, and S. Weiss, Science 255: 1707-1710, 1992). We instituted primary cultures of such progenitor cells from forebrains of newborn rat pups in an epidermal growth factor-containing medium free of serum in order to study the development of astrocytes in culture. At 4-6 days, primary cultures consisted of floating clusters of proliferating cells which expressed nestin, a marker for neuroepithelial progenitor cells, the ganglioside GD3, and vimentin. When clusters were transferred to polylysine-coated dishes, cells attached to the substrate and began to express antigens characteristic of particular differentiated neurons, astrocytes, or oligodendrocytes within 2 weeks. In 4- to 6-week-old secondary cultures, levels of vimentin expression appeared to decrease within maturing astrocytes which had increased levels of glial fibrillary acidic protein. These results suggest that multipotential epidermal growth factor-progenitor cells can give rise to both neurons and macroglia of the adult central nervous system, and that maturation of the astrocytes in vitro may be occurring in a pattern similar to that seen in vivo. Furthermore, no glial fibrillary acidic protein-positive cells expressed the A2B5 antigen in the same cell indicating an absence of type-2 astrocytes.

Study Objective: To determine whether elimination of intraoperative dextrose-containing infusions affects post-cardiopulmonary bypass hyperglycemia in pediatric patients., Design: Randomized, unblinded, saline-controlled study of perioperative glucose infusions in children undergoing cardiac surgery., Setting: Cardiac surgery suite and pediatric intensive care unit (ICU) of a university medical center., Patients: 33 consecutive, nondiabetic children undergoing cardiac surgery with deep hypothermia over an 8-month period., Interventions: Group A (n = 16) received only normal saline infusions intraoperatively, and Group B (n = 17) received 5% dextrose and lactated Ringer's solution exclusively. Blood glucose was sampled immediately after induction of anesthesia, prior to cardiopulmonary bypass (CPB), after separation from CPB, on arrival in the ICU, and the morning of the first postoperative day. Data were analyzed using Student's t-test for independent samples, paired t-test, and analysis of variance, with p < 0.05 considered significant., Measurements and Main Results: Although moderate elevations in blood glucose (mean less than 165 mg/dl) after CPB were present in Group A, significantly higher levels (mean greater than 250 mg/dl) were noted in Group B. No children were hypoglycemic (glucose less than 40 mg/dl). Glucose levels were normal in both groups on the morning of the first postoperative day despite patients' having received continuous dextrose infusions in the ICU and the presumed stress of emergence from anesthesia., Conclusions: Extreme postbypass hyperglycemia can be controlled by eliminating intraoperative dextrose infusions. Hypoglycemia, an unlikely event, can easily be avoided by regular blood sampling, which is facilitated by the routine placement of arterial catheters.

Recent evidence suggests that interleukin (IL)-1 and tumor necrosis factor (TNF) may play a role in astrogliosis following injury to the CNS. The short-term biochemical effects of these immune-related cytokines were determined on cultured rat polygonal and process-bearing astrocytes. Both IL-1 and TNF stimulated the rate of thymidine incorporation in polygonal astrocytes up to 137% and 215%, respectively, over the level observed in untreated controls. By contrast, thymidine incorporation was relatively unaffected by these cytokines in process-bearing astrocytes. The cytokines did not significantly affect the level of glial fibrillary acidic protein (GFAP) within polygonal astrocytes, even though they appeared to downregulate the expression of GFAP mRNA by as much as 62%. Both cytokines increased the intracellular expression of transferrin (Tf) within some polygonal astrocytes. In untreated control cultures, fewer than than 2% of polygonal astrocytes were immunoreactive for Tf. By contrast, approximately 30% of polygonal astrocytes treated with IL-1 or TNF-alpha became strongly immunoreactive for Tf. Neither IL-2 nor a number of other known growth factors appeared to alter the level of immunoreactive Tf in these cells. Process-bearing astrocytes were negative for Tf, regardless of the treatment used. Northern blot analysis demonstrated that the level of Tf mRNA in cultures of polygonal astrocytes increased 148% above the level observed in untreated controls following treatment with either IL-1 or TNF, whereas no change was observed following treatment with IL-2. These results suggest that increased levels of particular cytokines known to be present in injured CNS can produce pronounced biochemical alterations within a subtype of cultured astrocytes.

There is a significant incidence of inguinal hernia in premature infants and the optimal timing of repair is controversial. A high rate of postoperative respiratory complications has been reported in this group. In this study, the records of 47 premature infants (mean gestational age, 30.3 weeks) who underwent herniorrhaphy while still in the neonatal intensive care unit were reviewed in an effort to define those conditions that are independent risk factors for complications. Forty-three percent of infants had complications, including postoperative assisted ventilation (34%), episodes of apnea and/or bradycardia (23%), emesis and cyanosis with first feeding (6%), and requirement for postoperative reintubation (4%). Although low gestational age and postconceptual age at operation, low birth weight for gestational age, and preoperative ventilatory assistance were significantly associated with postoperative complications, only a history of respiratory distress syndrome/bronchopulmonary dysplasia (odds ratio 2.3), a history of patent ductus arteriosus (odds ratio 2.5), and low absolute weight at operation (odds ratio 3.5 for 1,000-g decrease) were independent risk factors for postoperative complication. Despite previous reports citing postconceptual age as the factor having the greatest impact on postoperative complications, these results indicate that a history of respiratory dysfunction and size at operation may be more important predictors of postoperative respiratory dysfunction in preterm infants.

Peripheral-type benzodiazepine binding sites (PTBBS) are markedly increased in the injured CNS. Astrocytes appear to be the primary cell type which express increased PTBBS. Because certain cytokines within the injured CNS are potent mitogens for astrocytes, we examined the effects of two such cytokines, interleukin (IL)-1 beta and tumor necrosis factor (TNF), on PTBBS in cultured astrocytes using [3H]Ro 5-4864 as the specific ligand. Purified cultures of either polygonal or process-bearing astrocytes were prepared from neonatal rat cerebral hemispheres. At a concentration of 1.8 nM, specific binding of the radioactive ligand to polygonal astrocytes reached equilibrium within 60 min and was half-maximal by 5-10 min. By contrast, specific binding to process-bearing astrocytes barely exceeded background levels. IL-1 and TNF increased PTBBS within polygonal astrocytes in both dose- and time-dependent manners. At 10-50 ng/ml, IL-1 beta and TNF-alpha elevated [3H]Ro 5-4864 binding in polygonal astrocyte cultures 65 and 87%, respectively, above the level in control cultures. However, no changes in PTBBS were seen within polygonal astrocytes after IL-2 treatment. Scatchard analysis of saturation binding experiments suggested that the increase in PTBBS promoted by TNF was due to an increased number of binding sites present in polygonal astrocytes and not due to an increase in receptor affinity. Binding data suggested that PTBBS within cultures of process-bearing astrocytes were virtually absent irrespective of the treatment. These in vitro data suggest that certain cytokines found in the injured brain may be involved in up-regulating PTBBS within a particular subtype of astrocyte.

During embryonic development, spinal motor neurons require muscle-derived trophic factors for their survival and growth. We have recently isolated a protein from muscle that is not laminin but that still stimulates neurite outgrowth from embryonic neurons in culture. In the present study, we investigated whether this protein, which we refer to as muscle-derived neurite-promoting factor (NPF), could also promote the survival and growth of motor neurons in culture. Spinal motor neurons were isolated from 6-day-old chicken embryos by a metrizamide step-gradient centrifugation protocol. Most large cells (putative motor neurons) were found in the upper metrizamide fraction (0%-6.8% interface; fraction I). Motor neurons were identified by increased specific activity of choline acetyltransferase (CAT) and by their propensity to transport retrogradely either wheat germ agglutinin-horseradish peroxidase or the fluorescent dye, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine per chlorate (diI), when those substances were injected into the target field. Labeled motor neurons were 2.6-fold enriched in fraction I and the specific CAT activity was 4.4-fold increased in fraction I as compared to unfractionated cells. When motor neurons were grown on muscle-derived NPF, the protein supported the survival of at least 21% of the neurons for as long as 6 days in culture. The protein showed no significant effect on either CAT specific activity or on high-affinity choline uptake by neurons. There was a substantial increase from 21% to 38% of the survival of motor neurons when a combination of muscle-derived NPF and laminin was used as the substrate. Muscle-derived NPF also promoted the survival of sensory neurons and sympathetic neurons in culture. Our results demonstrate that a neurite-promoting protein derived from muscle promotes both the survival and the outgrowth of neurites from cultured spinal motor neurons as well as from sensory and sympathetic neurons.

To assess the oxidative metabolism of glial cells, we visualized mitochondrial malate dehydrogenase (mMDH) in purified cultures of neonatal rat polygonal and process-bearing astrocytes as well as in oligodendrocytes, using indirect immunofluorescence. Double immunofluorescent localization of rabbit anti-mMDH and either mouse monoclonal antiglial fibrillary acidic protein or anti-myelin basic protein demonstrated that both process-bearing astrocytes and oligodendrocytes showed uniformly intense anti-mMDH immunoreactivity in their cell bodies. However, immunoreactivity to mMDH among polygonal astrocytes varied from very weakly positive to intensely positive. Experiments with rhodamine 123, a mitochondrion-specific fluorochrome, indicated that polygonal astrocytes contain relatively similar numbers of mitochondria; this suggested that the variable intensities of anti-mMDH immunoreactivity observed did not result from differences in mitochondrial numbers. In cultures of polygonal astrocytes maintained in a chemically defined medium containing growth factors and hormones, or in complete culture medium containing 1mM N6, O2-dibutyryl adenosine 3',5'-cyclic phosphate, the resultant stellate astrocytes still showed their original variable levels of anti-mMDH immunoreactivity. This suggested that the mMDH distribution pattern did not depend on the degree of morphological differentiation. Furthermore, cultures of polygonal astrocytes isolated from four specific regions of neonatal rat brain showed variable but reproducible profiles of anti-mMDH immunoreactivity. Our results suggest that there may be an appreciable range in the level of oxidative metabolism among individual polygonal astrocytes in culture.

Transferrin accumulates within neurons of the developing nervous system of humans, sheep, pigs and chickens. To assess the relationship of this accumulation with the ontogeny of oxidative metabolism, we studied the immunocytochemical localization of transferrin (Tf) and the mitochondrial form of malate dehydrogenase (mMDH) in developing neural tissues by the peroxidase-antiperoxidase method. Rabbit anti-rat Tf was obtained commercially and gave a single band of reaction product (MW = 80 kd) on Western blots. Antibodies to porcine heart mMDH were elicited in a rabbit. Western blot analysis showed that this anti-porcine mMDH antibody reacted with the mMDH from porcine, rat or avian tissue but not with the cytosolic MDH from pigs. Tf was first detected in rat brain neurons at about the 18th embryonic day and reached a peak at about the 6th postnatal day. All neurons were immunoreactive with large neurons throughout the brain showing a strong reaction for Tf. From this time onward, the level in brain neurons gradually decreased until adulthood. However, Tf immunoreactivity still remained strongly evident in capillary endothelial cells. The localization of Tf within rat spinal cord neurons peaked as early as the 1st postnatal day and remained elevated to the 6th postnatal day. By contrast, reactivity for Tf within dorsal root ganglia neurons was intense as early as the 18th embryonic day and diminished only gradually. Mitochondrial MDH, a marker for oxidative metabolism, appeared to reach a peak after the crest of intraneuronal Tf had been observed. For example, brain and spinal cord MDH immunoreactivity increased with intense staining in the cell bodies and fibers of neurons from the 6th to the 13th postnatal day; immunoreactivity gradually diminished into adulthood. The gradient of reactivity was low in some areas of the brain but more intense in areas containing large neuronal cell bodies such as the red nucleus. This occurred after the peak of intraneuronal Tf at day 6 and suggested a precursor-product relationship. By contrast, immunoreactivity for neuron-specific enolase, a glycolytic enzyme, showed a developmental pattern that differed from either Tf or MDH in that reactivity appeared later in development and was less intense. These data suggest that as cerebral metabolic rates begin to increase as early as 5-6 days after birth in the rat, an increase in mMDH occurs coincident with the onset of oxidative metabolism. Furthermore, this rise in intraneuronal mMDH follows the peak of intraneuronal Tf and suggests that Tf supplies the iron required for the synthesis of other mitochondrial ferroproteins.

Transferrin is a growth-promoting plasma protein which is known to occur within developing neurons. Since little information exists on the process by which transferrin is internalized by neurons, we studied this process using dissociated embryonic chicken dorsal root ganglion neurons in culture. Cultured dorsal root ganglion neurons were incubated in the presence of 3.75 nM (125)I-transferrin at 37°C, the cultures were extensively washed, the neurons were solubilized in a Triton-containing buffer and internalized (125)I-transferrin was quantified with a gamma counter. (125)I-transferrin was internalized in a linear fashion for at least 60 min, and this uptake was abolished by the presence of 1.25 μM unlabeled transferrin. No competition for the uptake of (125)I-transferrin was observed in the presence of 1.25 μM ovalbumin, cytochrome c, hemoglobin, insulin, horseradish peroxidase, aldolase or the carboxyl-terminal fragment ('half-site') of transferrin. By contrast, uptake was inhibited by approximately 50% in the presence of the ammo-terminal fragment ('half-site') of transferrin (1.25 μM) or in the presence of concanavalin A (1.25 μM). The binding of transferrin conjugated to fluorescein isothiocyanate to neurons at 4°C and its subsequent internalization at 37°C was demonstrated by fluorescence microscopy of unfixed cells following incubation of the neurons in the presence of the fluorescently labeled protein. Furthermore, the transferrin receptors were visualized immunocytochemically on the surface membranes of dorsal root ganglion neurons using rabbit antibodies directed against transferrin receptors from chicken reticulocytes. From these data, we conclude that transferrin is internalized by neurons via receptor-mediated endocytosis, and suggest that this protein may serve an important role in the development and survival of dorsal root ganglion neurons., (Copyright © 1985. Published by Elsevier Ltd.)

Transferrin is one of several serum proteins localized within neurons during development of the nervous system. The expression of transferrin receptors appears to precede the active accumulation of transferrin by neurons. The first cells immunoreactive for transferrin appear adjacent to the ventricles or to the central canal of the spinal cord. These cells then appear to migrate from this site. These neurons become progressively more immunoreactive for transferrin, attain a peak of reactivity and then lose their reaction to antitransferrin antibodies. Thus, a "window" of transferrin immunoreactivity is found. As neurons lose their reactivity to antitransferrin antibodies, glia and the walls of capillaries become positive. In the rat nervous system, the gradual decrease in intraneuronal transferrin is accompanied by an increase in mitochondrial malate dehydrogenase, an enzyme of the tricarboxylic acid cycle. Thus, the accumulation of transferrin appears to closely precede the ontogeny of oxidative metabolism in the brain. As transferrin appears transiently in all neurons, this protein may be involved in a number of other important developmental events such as the expression of dopamine D2 receptors and the period of "programmed" cell death in the spinal cord.

Esophageal stricture commonly occurs in patients with epidermolysis bullosa dystrophica recessive (EBDR), but esophageal replacement is considered a high risk procedure because of limited exposure of the airway, malnutrition, and postoperative skin bullae to secondary infection. Recent innovations in care, including preoperative parenteral nutrition, topical care for bullae and skin ulceration, fiberoptic tracheal intubation, electrocardiogram monitoring with metallic pacer leads, and an overall concern to protect the fragile intact skin, have improved the results of esophageal replacement. Utilizing these adjunctive measures, ileocolonic substernal interposition has been successfully performed in a 26-yr-old male and a 19-yr-old female at our institution. Despite cervical anastomotic stricture requiring resection in one, and an obstructive cervical esophageal bullous developing acutely 5 yr after operation in the second, both patients now gum or chew a solid diet. The first patient also benefited from total esophagectomy for squamous dysplasia detected at the time of esophageal replacement. Multidisciplinary management by the surgeon, gastroenterologist, anesthesiologist, and dermatologist makes esophageal replacement available for younger patients with epidermolysis bullosa dystrophica recessive and esophageal strictures.

A mytotrophic protein (sciatin) purified from chicken sciatic nerves has "trophic" or "maintenance" effects on cultured muscle. We have elicited a specific antiserum against purified sciatin in rabbits. Using this antiserum, we investigated the distribution of sciatin in embryonic and adult chicken tissues by an unlabeled peroxidase-an-tiperoxidase method at the light microscopic level. The antiserum stained adult chicken neural tissues in situ and cultured embryonic chick neurons. Staining was intense in the cell bodies of spinal cord neurons and the axoplasm of sciatic nerves. These was reaction product seen in the outer margins of myelin sheaths that corresponded to the Schwann cell cytoplasm. Cerebral cortical neurons were weakly stained by the antiserum. No staining was apparent in oligodendrocytes or astrocytes. Nonneural tissues, such as skeletal, smooth and cardiac muscle, kidney, and liver, were also unstained by the antiserum. Cultured spinal cord neurons, cerebral cortical neurons, and sensory neurons were stained immunocytochemically by the antiserum. There was no reaction product seen in the glial cells that are usually present in neuronal cultures or cultured cells from liver, kidney, skeletal muscle, smooth muscle, and cardiac muscle. Our results thus show that the myotrophic protein is localized in neuronal perikarya and their processes in vivo as well as in vitro.

Astroglia in cultures of dissociated neonatal rat optic nerves were studied by light microscopy, immunocytochemistry and electron microscopy to determine whether intermediate filaments play a role in defining the multipolar morphology of the mature astrocyte. Immature, polygonal astroblasts contained few glial filaments, in spite of exhibiting positive staining with antiserum against glial fibrillary acidic (GFA) protein. Microtubules were the most prominent cytoskeletal component at early stages of cytodifferentiation, but these were progressively reduced in number at later intervals and were gradually replaced by intermediate filaments. These observations suggest that microtubules are involved in the initial establishment of cytoplasmic asymmetry and process development. Subsequently, glial filaments may play a role in maintaining and stabilizing the overall geometry of the mature astrocyte.

1. Primary cultures of dissociated embryonic chicken skeletal muscle cells provide an ideal model for investigating the effects of growth factors such as Tf because these cells undergo a highly integrated pattern of differentiation and maturation. 2. The trophic effects of a growth factor such as Tf can be assessed on muscle cultures by the determination of such parameters as acetylcholinesterase and acetylcholine receptors. These proteins are specific to the cultured myotubes, appear in high levels following fusion of myoblasts into myotubes, and are relatively easy to assay. 3. Tf and other growth factors are internalized by a receptor-mediated mechanism (see Trowbridge et al. and Seligman and Allen, this volume). These growth factors can be localized to specific tissues by immunocytochemistry at the light or electron microscopic level. This information on cellular distribution could be very useful in assessing the pattern of growth and differentiation with regard to the particular growth factor under study.

The volume and pressure of a tracheal tube cuff inflated with air increases during nitrous oxide anaesthesia. The study was designed to investigate the changes of tracheal tube cuff pressure during nitrous oxide anaesthesia following inflation of the cuff with air or saline in 10 mongrel dogs who were anaesthetised with nitrous oxide and their lungs artificially ventilated. At the end of 6 hours there was no change in cuff pressure when saline had been used for inflation, but was six times the initial pressure when air had been used. On microscopic examination of the trachea, only the air group had glandular inflammation, dilatation and destruction. Therefore, it appears that if saline is used to inflate tracheal tube cuffs, there will not be an increase in cuff volume and pressure during nitrous oxide anaesthesia.