Photodynamic therapy of red and blue lights on Malassezia pachydermatis: an in vitro study.

In veterinary medicine, infection caused by Malassezia pachydermatis is spreading and necessity of alternative treatment is emphasized. Photodynamic therapy (PDT) is therapeutic method using specific spectrum of light with photosensitizer. In this study, applying PDT not only using red light which is used in human medicine commonly, but also using blue light into skin infection causative microorganism with photosensitizer, confirm the effect of PDT and possibility of being an alternative treatment. Four isolates of M. pachyderematis were collected from canine skin and used into this study. Light emitting diode with 495 nm, 625 nm spectrum was applied, and final concentration of δ-aminolevulinic acid (ALA), which is used as a photosensitizer, was adjusted into 20%. To confirm effectiveness of PDT, the number of colony forming unit was checked and variation of optical density values was measured. Antifungal effect of PDT on both spectrums was presented in all condition, and it makes best result when using blue light applied with ALA. Through outcome of this study, PDT using light in 465 nm, 625 nm wavelength combinations with ALA can interrupt proliferation of M. pachydermatis considerably. In consequence, PDT can be alterative treatment of canine Malassezia infection.

Late Radiological and Clinical Outcomes of Traumatic Thoracic Aortic Injury Managed with Thoracic Endovascular Aortic Repair.

PURPOSE: Patients treated with thoracic endovascular aortic repair (TEVAR) for traumatic thoracic aortic injury (TTAI) are often young and data on long-term durability of this treatment is not widely documented. The aims of this study were to report the New Zealand (NZ) national experience of TEVAR and to assess the durability of late outcomes and radiological follow-up of patients treated for TTAI. METHODS: Consecutive patients treated with TEVAR during a 12-year period from all tertiary centers in NZ were included. Early (30-day), late survival and radiological imaging data were recorded to document late graft-related complications and re-interventions. RESULTS: 88 patients with a median (range) age of 35 (15-87) year and 63 (71.6 %) males were included. Eleven patients (12.5 %) died within 30 days, of which three were aortic related deaths. The median (range) follow-up was 76.3 (0.3-164.6) months. Six (7.8 %) patients died during the follow-up period due to non-aortic-related causes. Nine (11.5 %) patients were lost to follow-up of which three emigrated overseas. Of those on surveillance, two patients required TEVAR re-intervention to previously treated aortic segments; one for a type 1b endoleak and the other for a symptomatic pseudo-coarctation. Both were treated successfully with a TEVAR. CONCLUSIONS: This multicenter study suggests that TEVAR is a durable option for treatment of traumatic thoracic aortic injury. Although, stent graft complications were uncommon, but when it occurred, it leads to re-intervention. Further radiological follow-up is required particularly in young patient to document late aortic/stent complications.

Association of rheumatic diseases with symptom severity, quality of life, and treatment outcome in patients with fibromyalgia.

OBJECTIVES: To evaluate the frequency of rheumatic diseases and their association with symptom severity, quality of life (QoL), and treatment outcome in patients with fibromyalgia (FM). METHOD: Our study contained 536 FM patients who completed a brief, interdisciplinary fibromyalgia treatment programme (FTP) at our institution, with emphasis on cognitive behavioural therapy (CBT). The Fibromyalgia Impact Questionnaire (FIQ) and the 36-item Short Form Health Status Questionnaire (SF-36) were completed at initial evaluation and at 6 and 12 months after the FTP. The presence of inflammatory rheumatic disease (IRD) was determined by physician diagnoses. A two-sample t-test and multivariate linear regression analyses were performed to compare the rheumatic and non-rheumatic groups. RESULTS: Thirty-six patients (6.7%) had documented IRD. At baseline, the rheumatic group had poorer scores in SF-36 physical functioning (p = 0.02), pain index (p = 0.01), and physical component summary (p = 0.009) than the non-rheumatic group. After treatment, both groups tended to improve; however, the rheumatic group had significantly less improvement on the FIQ subscales in pain (p = 0.01) and missed work days (p = 0.01), as well as in the SF-36 physical functioning (p = 0.01), pain index (p = 0.049), and physical component summary (p = 0.049) compared with the non-rheumatic group. CONCLUSIONS: The frequency of rheumatic diseases in patients with FM seen at FTP was 6.7%. FM patients with rheumatic diseases were found to have worse SF-36-assessed pain and physical health and less improvement in these measures following treatment from FTP than patients without rheumatic diseases. FM patients with rheumatic disease may require additional intervention to address underlying rheumatic disease-related limitations.

Endoscopic Treatment for Persistent Hematospermia: A Novel Technique Using a Holmium Laser.

PURPOSE: We present our endoscopic technique for treating ejaculatory duct and seminal vesicle diseases with a holmium laser. MATERIALS AND METHODS: Fifteen patients with persistent hematospermia were enrolled in this study from June 2007 to April 2014. All patients had failed medical treatments. All patients were evaluated with transrectal ultrasound and pelvic computed tomography or magnetic resonance imaging. We performed endoscopic treatment with a semi-rigid ureteroscope after dilation using a guidewire and ureteral serial dilator. A holmium laser was used to incise the obstructed ejaculatory duct, coagulate hemorrhagic mucosa, and fragment stones in the ejaculatory duct or seminal vesicles. Stones were removed using a basket and forceps. RESULTS: The mean duration of hematospermia was 30.6 months. Mean patient age was 45.3 years. The mean serum levels of prostate-specific antigen and testosterone were 1.36 and 4.95 ng/mL, respectively. No operative complications were encountered. Mean operative time was 35.4 min. Seven patients had ejaculatory duct or seminal vesicle stones, which were subsequently determined to be carbonate apatite, mucin, struvite, and calcium oxalate dehydrate stones. Mean duration of follow-up was 32.1 months. Although two patients showed recurrent hematospermia 11 and 12 months after the operation, hematospermia resolved in 13 patients (86.7%). The infertile patient showed an improved semen finding and had a successful pregnancy. CONCLUSION: Endoscopic treatment using a holmium laser is minimally invasive and was effective for treating ejaculatory duct and seminal vesicle diseases, which are the main cause of hematospermia.

The utility of a 'non-significant' coronary angiogram.

BACKGROUND: Coronary angiography is the gold standard for assessing coronary artery disease (CAD). In many patients with chest pain, no or mild CAD (< 50% stenosis) is found. It is uncertain whether this 'non-significant' result influences management and outcomes. We reviewed characteristics and outcomes in a contemporary cohort of chest pain referrals who had mild or absent CAD on coronary angiography. METHOD: All patients undergoing coronary angiography at Auckland City Hospital during July 2010-October 2011 were reviewed (n = 2983). Of these, 12.3% (n = 366) underwent coronary angiography for evaluation of chest pain and were found to have absent or mild CAD. These patients were followed up for 2.3 ± 0.6 years. RESULTS: Mean age was 60.0 ± 12.3 years, 56.1% were female. The ECG was abnormal in 55.0% of patients. Stress testing for inducible ischaemia was undertaken in 40.7% of patients and was abnormal in 57.7%. Following angiography, 43.2% had no changes to cardiac medications. Additional drug therapy (aspirin, statin, beta-blockers, ACE-inhibitor) was commenced in around 14.2-22.1% of cases. These drugs were discontinued in 4.1-8.2% of patients. Rates of major adverse cardiovascular events and readmissions with chest pain were 0.3% (1) and 1.9% (7) respectively at 30 days, and 1.9% (7) and 6.0% (22) at 1 year. CONCLUSION: Although even non-obstructive atheroma may justify medical therapy to limit disease progression, our findings may suggest that in these cases, invasive coronary angiography, may not lead to the patient/physician reassurance justified by historical data.

Gait patterns of patients with inclusion body myositis.

Inclusion body myositis (IBM) is a progressive, inflammatory muscle disease that is known to cause quadriceps weakness and knee buckling during gait. This is the first known report of gait characteristics in patients with IBM. Nine subjects with IBM and quadriceps weakness underwent gait analysis and quantitative strength testing. A wide range of strength and gait abilities were present in the subject group. Subjects with stronger knee extensors exhibited nearly normal sagittal knee kinematics and kinetics. As quadriceps strength decreased, kinematic and kinetic patterns were increasingly abnormal. Exceptions to this pattern could be explained by examining strength at adjacent joints. Gait analysis and strength testing is a helpful tool in evaluating the functional status of this population and aiding in determination of the needs for interventions such as assistive devices.

Measurement of transepidermal water loss from clipped and unclipped anatomical sites on the dog.

OBJECTIVES: To establish a standardised clipping method for the measurement of transepidermal water loss (TEWL) with a VapoMeter in Beagle dogs and to identify the optimal anatomical site for TEWL measurement. PROCEDURE: TEWL values obtained from skin sites on five healthy Beagles clipped using two different blade angles (standard vs non-standard) were compared. TEWL values for 48 h were also obtained from seven different anatomical sites that had differing hair density. The hair was clipped in the intensively haired anatomical sites (head, lower and upper back and tail), but not clipped in the sparsely haired sites (ear, inguinal region, footpad). RESULTS: The TEWL values for the standard and non-standard clipping sites were 6.3 +/- 1.31 and 27.2 +/- 1.11 g/h/m(2), respectively. We found the upper back among the clipped sites was the most appropriate site for TEWL measurement over 48 h after clipping, whereas among the unclipped sites the ear was the most appropriate, because the TEWL values from those anatomical sites had the least fluctuation and were less affected by movement. CONCLUSION: The clipping method and anatomical site should be standardised in order to minimise the experimental variation in TEWL measurement in dogs.

Pinusolide and 15-methoxypinusolidic acid attenuate the neurotoxic effect of staurosporine in primary cultures of rat cortical cells.

BACKGROUND AND PURPOSE: Apoptosis is a fundamental process required for neuronal development but also occurs in most of the common neurodegenerative disorders. In an attempt to obtain an anti-apoptotic neuroprotective compound from natural products, we isolated the diterpenoids, pinusolide and 15-MPA, from B. orientalis and investigated their neuroprotective activity against staurosporine (STS) -induced neuronal apoptosis. In addition, we determined the anti-apoptotic mechanism of these compounds in rat cortical cells. EXPERIMENTAL APPROACH: Primary cultures of rat cortical cells injured by STS were used as an in vitro assay system. Cells were pretreated with pinusolide or 15-MPA before exposure to STS. Anti-apoptotic activities were evaluated by the measurement of cytoplasmic condensation and nuclear fragmentation. The levels of cellular peroxide, malondialdehyde (MDA) and [Ca(2+)]i, as well as the activities of superoxide dismutase (SOD) and caspase-3/7, were measured. KEY RESULTS: Pinusolide and 15-MPA, at a concentration of 5.0 ìM, reduced the condensed nuclei and rise in [Ca(2+)]i that accompanies apoptosis induced by 100 nM STS. Pinusolide and 15-MPA also protected the cellular activity of SOD, an antioxidative enzyme reduced by STS insult. Furthermore, the overproduction of reactive oxygen species and lipid peroxidation induced by STS was significantly reduced in pinusolide and 15-MPA treated cells. In addition, pinusolide and 15-MPA inhibited STS-induced caspase-3/7 activation. CONCLUSIONS AND IMPLICATIONS: These results show that pinusolide and 15-MPA protect neuronal cells from STS-induced apoptosis, probably by preventing the increase in [Ca(2+)]i and cellular oxidation caused by STS, and indicate that they could be used to treat neurodegenerative diseases.

Temporospatial sequence of cellular events associated with etoposide-induced neuronal cell death: role of antiapoptotic protein Bcl-X(L).

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.

Overexpression of calbindin-D28K induces neurite outgrowth in dopaminergic neuronal cells via activation of p38 MAPK.

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.

MPP(+) downregulates mitochondrially encoded gene transcripts and their activities in dopaminergic neuronal cells: protective role of Bcl-2.

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.

Cleavage of Bax is mediated by caspase-dependent or -independent calpain activation in dopaminergic neuronal cells: protective role of Bcl-2.

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.

Intracellular calcium mobilization induces immediate early gene pip92 via Src and mitogen-activated protein kinase in immortalized hippocampal 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.

Role of tumor necrosis factor-alpha in neuronal and glial apoptosis after spinal cord injury.

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.

Preparation of monoclonal antibody against ginsenoside Rf and its enzyme immunoassay.

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.

Rapid increase in immunoreactivity to GFAP in astrocytes in vitro induced by acidic pH is mediated by calcium influx and calpain I.

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.

Correlation between structure of Bcl-2 and its inhibitory function of JNK and caspase activity in dopaminergic neuronal apoptosis.

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.