The essential and downstream common proteins of amyotrophic lateral sclerosis: A protein-protein interaction network analysis.

Amyotrophic Lateral Sclerosis (ALS) is a devastative neurodegenerative disease characterized by selective loss of motoneurons. While several breakthroughs have been made in identifying ALS genetic defects, the detailed molecular mechanisms are still unclear. These genetic defects involve in numerous biological processes, which converge to a common destiny: motoneuron degeneration. In addition, the common comorbid Frontotemporal Dementia (FTD) further complicates the investigation of ALS etiology. In this study, we aimed to explore the protein-protein interaction network built on known ALS-causative genes to identify essential proteins and common downstream proteins between classical ALS and ALS+FTD (classical ALS + ALS/FTD) groups. The results suggest that classical ALS and ALS+FTD share similar essential protein set (VCP, FUS, TDP-43 and hnRNPA1) but have distinctive functional enrichment profiles. Thus, disruptions to these essential proteins might cause motoneuron susceptible to cellular stresses and eventually vulnerable to proteinopathies. Moreover, we identified a common downstream protein, ubiquitin-C, extensively interconnected with ALS-causative proteins (22 out of 24) which was not linked to ALS previously. Our in silico approach provides the computational background for identifying ALS therapeutic targets, and points out the potential downstream common ground of ALS-causative mutations.

Distribution of lysosome-associated membrane proteins-1 and -2, and cathepsin D in eosinophilic granular bodies: possible relationship to cyst development in pilocytic astrocytomas.

Pilocytic astrocytomas are usually cystic; cyst formation within these tumours may result in increased intracranial pressure, due to the effect of their mass, and contribute to cerebral damage. Eosinophilic granular bodies (EGBs) are produced abundantly in pilocytic astrocytomas but their role in disease progression remains unknown. Immunohistochemistry studies showed EGBs to exhibit pronounced reactivity to antibodies against lysosome-associated membrane proteins (LAMP)-1 and LAMP-2, and the lysosomal enzyme cathepsin D. Both LAMP-1 and LAMP-2 showed peripheral rim and granular staining patterns. The EGBs were scattered widely across cysts and, where EGBs aggregated in clusters, were usually close to areas of fluid in the cysts. Most EGBs had nuclei either attached or close by, indicating that the EGBs may be derived from anucleated astrocytes. The results suggest that EGBs, together with other factors, may play a role in the development of cysts in pilocytic astrocytomas.

Discrimination between Sjögren's and non-Sjögren's sicca syndrome by sialoscintigraphy and antibodies against alpha-fodrin and Ro/La autoantigens.

Both Sjögren's syndrome (SS) and non-Sjögren's syndrome (NSS) can present with the sicca symptoms of dry eyes and a dry mouth but they are distinct pathological entities that require diagnostic discrimination. This study included 82 sicca syndrome patients and examined the ability of sialoscintigraphy and antibodies against the autoantigens alpha-fodrin, Ro and La to discriminate between SS and NSS. A total of 30.8% of SS patients compared with 58.8% of NSS patients were alpha-fodrin positive. The prevalence of Ro positivity was 69.4% for SS patients compared with 0% for NSS patients. The prevalence of La positivity was 52.4% for SS compared with 0% for NSS patients. Sialoscintigraphy showed that more NSS patients had grade III salivary gland impairment compared with SS patients (64.7% versus 19.4%). These data suggest that using sialoscintigraphy in combination with measuring the levels of serum alpha-fodrin, Ro and La might be useful for SS and NSS discrimination.

Synergic CSE1L/CAS, TNFR-1, and p53 apoptotic pathways in combined interferon-gamma/adriamycin-induced apoptosis of Hep G2 hepatoma cells.

Many cancers are chemotherapy-resistant. Chemotherapy combined with immunotherapy offers a potential avenue for the treatment of chemotherapy-resistant cancers. In this study, we investigated the apoptotic pathways induced by combined interferon-gamma/adriamycin treatment in Hep G2 cells. Our data showed that Hep G2 cells treated with combined interferon-gamma/adriamycin enhanced cell apoptosis in comparison with that of cells treated with adriamycin. Interferon-y increased TNFR-1, CSE1L/CAS (cellular apoptosis susceptibility protein), Bax, and Bad levels. Adriamycin increased p53 and Bax, but not TNFR- 1 and CAS levels. Interferon-y did not increase p53 accumulation; nevertheless it enhanced adriamycin-induced p53 accumulation. Overexpression of IRF-1 augmented the combined interferon-gamma/adriamycin-induced p53 accumulation. Interferon-gamma co-treatment increased the stability of p53 protein induced by adriamycin. Our data suggest that TNF-gamma may greatly enhance the combined interferon-gamma/chemotherapeutic drug-induced apoptosis of cancers. Our findings also indicate that CAS, TN-FR-1, p53, Bax, and Bad may be the targets for the interferon-y-based chemo-immunotherapy of the chemotherapy-resistant cancers.

In vitro sacral cord preparation and motoneuron recording from adult mice.

We report the development of an intracellular recording technique for adult mouse motoneurons in sacral spinal cord. Based on a similar preparation for adult rat, we modified the cord preparation solution and filled the sharp electrode with a solution that has physiological osmolarity and pH. The viability of the preparation was examined by recording root reflexes. Short-latency reflexes mediated through monosynaptic transmission between S1 and S3 ventral root were reliably produced by dorsal root electrical stimuli and were stably recorded for more than eight hours. Long-lasting potentiation of the root reflex was observed by bath application of methoxamine, a noradrenergic alpha1 receptor agonist. Bath application of strychnine and picrotoxin, antagonists for glycine and GABA(A) receptors respectively, unmasked long-lasting reflexes that may contain polysynaptic components. In addition, on the background of strychnine and picrotoxin, adding methoxamine induced spontaneous ventral root activity. For intracellular recording, the motoneurons could be reliably penetrated and held for up to 30 min. In all 16 motoneurons recorded, resting membrane potential, input resistance, action potentials and repetitive firing were comparable to those of rat motoneurons. Thus, this preparation is viable and provides a new method for combined electrophysiological and genetic studies of the adult mouse spinal cord.

CSE1/CAS overexpression inhibits the tumorigenicity of HT-29 colon cancer cells.

We previously reported that CSE1/CAS (CAS) overexpression in HT-29 human colon cancer cells enhances the formation of the E-cadherin/beta-catenin complex, stimulates intercellular junction formation, and stimulates polarization of HT-29 cells. Since both E-cadherin/beta-catenin interaction and epithelial cell polarization are critically related to the tumorigenicity of carcinoma cells, we studied the role of CAS in the tumorigenicity of HT-29 colon carcinoma cells. CAS overexpression in HT-29 cells decreased the intercellular gaps and increased the compactness of cell colonies. Our results show that CAS expression inhibited migration and growth of HT-29 cancer cells. In the soft agar anchorage-independent growth assays, CAS overexpression inhibited the colony size of HT-29 cells by 74%, and inhibited colony formation number of HT-29 cells by 38%. CAS overexpression also inhibited the growth of HT-29 cells in nude mice. Our results indicate that CAS inhibits the tumorigenicity of HT-29 human colon cancer cells and, thus, it is worthwhile to further study CAS's possible role in the control of human colon cancer.

Influence of active dendritic currents on input-output processing in spinal motoneurons in vivo.

The extensive dendritic tree of the adult spinal motoneuron generates a powerful persistent inward current (PIC). We investigated how this dendritic PIC influenced conversion of synaptic input to rhythmic firing. A linearly increasing, predominantly excitatory synaptic input was generated in triceps ankle extensor motoneurons by slow stretch (duration: 2-10 s) of the Achilles tendon in the decerebrate cat preparation. The firing pattern evoked by stretch was measured by injecting a steady current to depolarize the cell to threshold for firing. The effective synaptic current (I(N), the net synaptic current reaching the soma of the cell) evoked by stretch was measured during voltage clamp. Hyperpolarized holding potentials were used to minimize the activation of the dendritic PIC and thus estimate stretch-evoked I(N) for a passive dendritic tree (I(N,PASS)). Depolarized holding potentials that approximated the average membrane potential during rhythmic firing allowed strong activation of the dendritic PIC and thus resulted in marked enhancement of the total stretch-evoked I(N) (I(N,TOT)). The net effect of the dendritic PIC on the generation of rhythmic firing was assessed by plotting stretch-evoked firing (strong PIC activation) versus stretch-evoked I(N,PASS) (minimal PIC activation). The gain of this input-output function for the neuron (I-O(N)) was found to be ~2.7 times as high as for the standard injected frequency current (F-I) function in low-input conductance neurons. However, about halfway through the stretch, firing rate tended to become constant, resulting in a sharp saturation in I-O(N) that was not present in F-I. In addition, the gain of I-O(N) decreased sharply with increasing input conductance, resulting in much lower stretch-evoked firing rates in high-input conductance cells. All three of these phenomena (high initial gain, saturation, and differences in low- and high-input conductance cells) were also readily apparent in the differences between stretch-evoked I(N,TOT) and I(N, PASS) and thus could be accounted for by the activation of the dendritic PIC. As a result, stretch-evoked I(N,TOT) and F-I provided an accurate prediction of the overall change in stretch-evoked firing. However, in about half of the low-input conductance cells, the rate of rise of firing in response to stretch was not smoothly graded but instead consisted of a rapid surge. Stretch-evoked I(N,TOT) was always smoothly graded. This suggests that although stretch-evoked I(N,TOT) can be used to predict the overall change in firing, prediction of the dynamics of firing may be less accurate.

Cerebellar input to magnocellular neurons in the red nucleus of the mouse: synaptic analysis in horizontal brain slices incorporating cerebello-rubral pathways.

We studied the synaptic input from the nucleus interpositus of the cerebellum to the magnocellular division of the red nucleus (RNm) in the mouse using combined electrophysiological and neuroanatomical methods. Whole-cell patch-clamp recordings were made from brain slices (125-150 microm) cut in a horizontal plane oriented to pass through both red nucleus and nucleus interpositus. Large cells that were visually selected and patched were injected with Lucifer Yellow and identified as RNm neurons. Using anterograde tracing from nucleus interpositus in vitro, we examined the course of interposito-rubral axons which are dispersed in the superior cerebellar peduncle. In vitro monosynaptic responses in RNm were elicited by an electrode array placed contralaterally in this pathway but near the midline. Mixed excitatory post-synaptic potentials (EPSPs)/inhibitory post-synaptic potentials (IPSPs) were observed in 48 RNm neurons. Excitatory components of the evoked potentials were studied after blocking inhibitory components with picrotoxin (100 microM) and strychnine (5 microM). All RNm neurons examined continued to show monosynaptic EPSPs after non-N-methyl-D-aspartate (NMDA) glutamate receptor components were blocked with 10 microM 6,7-dinitroquinoxaline-2,3-dione or 5 microM 2,3-dihydro-6-nitro-7-sulfamoyl-benzo(f)-quinoxaline (NBQX; n=12). The residual potentials were identified as NMDA receptor components since they (i) were blocked by the addition of the NMDA receptor antagonist, D,L-2-amino-5-phosphonovaleric acid (APV), (ii) were voltage-dependent, and (iii) were enhanced by Mg(2+) removal. Inhibitory components of the evoked potentials were studied after blocking excitatory components with NBQX and APV. Under these conditions, all RNm neurons studied continued to show IPSPs. Blockade of GABA(A) receptors reduced but did not eliminate the IPSPs. These were eliminated when GABA(A) receptor blockade was combined with strychnine to eliminate glycine components of the IPSPs. Thus, IPSPs evoked by midline stimulation of the superior cerebellar peduncle, while blocking alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and NMDA receptors, raise the possibility of direct inhibitory inputs to RNm from the cerebellum. In summary we propose that the special properties of the NMDA receptor components are considered important for the generation of RNm motor commands: their slow time course will contribute a steady driving force for sustained discharge and their voltage dependency will facilitate abrupt transitions from a resting state of quiescence to an active state of intense motor command generation.

Parvalbumin in respiratory neurons of the ventrolateral medulla of the adult rat.

A column of parvalbumin immunoreactive neurons is closely associated with the location of respiratory neurons in the ventrolateral medulla of the rat. The majority (66%) of bulbospinal neurons in the medullary ventral respiratory column (VRC) that were retrogradely labeled by tracer injections in the phrenic nucleus were also positive for parvalbumin. In contrast, only 18.8% of VRC neurons retrogradely labeled after a tracer injection in the VRC, also expressed parvalbumin. The average cross-sectional area of VRC neurons retrogradely labeled after VRC injections was 193.8 microm2 +/- 6.6 SE. These were significantly smaller than VRC parvalbumin neurons (271.9 microm2 +/- 12.3 SE). Parvalbumin neurons were found in the Bötzinger Complex, the rostral ventral respiratory group (VRG), and the caudal VRG, areas which all contribute to the bulbospinal projection. In contrast, parvalbumin neurons were sparse or absent in the preBötzinger Complex and in the vicinity of the retrotrapezoid nucleus, areas that have few bulbospinal projections. Parvalbumin was rarely colocalized within Neurokinin-1 receptor positive (NK1R) VRC neurons, which are found in the preBötzinger complex and in the anteroventral part of the rostral VRG. Parvalbumin neurons in the Bötzinger Complex and rostral VRG help define the rostrocaudal extent of these regions. The absence of parvalbumin neurons from the intervening preBötzinger complex also helps establish the boundaries of this region. Regional boundaries described in this manner are in good agreement with earlier physiological and anatomical studies. Taken together, the distributions of parvalbumin, NK1R and bulbospinal neurons suggest that the rostral VRG may be subdivided into distinct, anterodorsal, anteroventral, and posterior subdivisions.

IRF-1-mediated CAS expression enhances interferon-gamma-induced apoptosis of HT-29 colon adenocarcinoma cells.

The expression of CAS is reported to be upregulated in a variety of human tumor cells, and such expression correlates with the development of tumors. CAS also plays a role in apoptosis. We investigated whether CAS expression affects the susceptibility of tumor cells to IFN-gamma-induced apoptosis. Our data show that IFN-gamma treatment induces CAS expression in HT-29 tumor cells. IFN-gamma-induced gene expression is primarily mediated by the transcriptional factor, IRF-1. Our data show that IRF-1 mediates IFN-gamma-induced CAS expression. Transfection of HT-29 cells with CAS expression vector did not induce apoptosis of cells; nevertheless, CAS overexpression greatly enhanced IFN-gamma-induced apoptosis of cells. CPP32 is regarded as one of the central apoptosis executioner molecules. CAS overexpression enhances IFN-gamma-induced CPP32 expression. These results indicate that tumor cells highly expressing CAS may be more susceptible to apoptosis induced by reagents that are capable of inducing CAS expression. Thus, CAS may be a target for the elimination of tumors.

Aspirin inhibits matrix metalloproteinase-2 activity, increases E-cadherin production, and inhibits in vitro invasion of tumor cells.

Aspirin (acetylsalicylic acid) is a widely used anti-inflammatory drug. Recently, aspirin was shown to reduce the risk of development of cancer and mortality from it. Tumor metastasis is the most important cause of cancer death. The aim of the present study was to investigate if aspirin affects the invasion of cancer cells. Matrix metalloproteinases (MMPs) and cell adhesion molecules play important roles in the modulation of tumor invasion. Gelatin-based zymography assay showed that aspirin inhibited MMP-2 activity of SK-Hep-1 cancer cells. Matrigel-based chemoinvasion assay showed that aspirin inhibited in vitro invasion of SK-Hep-1 cancer cells. Aspirin treatment also increased the production of the cell adhesion molecule, E-cadherin, in Hep G2 cancer cells. Aspirin is a cyclooxygenase (COX) inhibitor. Treatment of cells with another COX inhibitor, sulindac, also inhibited MMP-2 activity and increased E-cadherin production of cells. These results indicate that aspirin can modulate both MMP-2 and E-cadherin production and therein may possess antimetastatic effect.

17 beta-estradiol inhibits tumor necrosis factor-alpha-induced nuclear factor-kappa B activation by increasing nuclear factor-kappa B p105 level in MCF-7 breast cancer cells.

Tumor necrosis factor-alpha (TNF-alpha) exerts many cytological effects on a wide range of cells. TNF-alpha can activate nuclear factor-kappa B (NF-kappa B). Activation of NF-kappa B by TNF-alpha mediates many functions of TNF-alpha. The NF-kappa B inhibitor, I kappa B alpha, negatively regulates the activity of NF-kappa B. In MCF-7 cells (an estrogen and TNF-alpha receptor positive cell line), treatment with 17 beta-estradiol (E(2)) inhibited TNF-alpha-induced NF-kappa B DNA binding activity in the gel retardation assays. But, the level of the I kappa B alpha and the TNF-alpha receptor, TNF-R1, were not obviously affected. The NF-kappa B precursor, NF-kappa B p105, has been shown to be associated with NF-kappa B in the cytoplasm and efficiently blocks its nuclear translocation and activation. Treatment of MCF-7 cells with E(2) increased the level of NF-kappa B p105 protein. The anti-estrogen, 4OH-tamoxifen, treatment inhibited E(2)-induced NF-kappa B p105 expression. Our findings indicate that NF-kappa B p105 plays a role in modulating the functions of TNF-alpha in the estrogen receptor positive breast cancer cells.

Methyl methanesulfonate and hydrogen peroxide differentially regulate p53 accumulation in hepatoblastoma cells.

Genotoxic chemicals not only damage cellular DNA, but may also induce cell apoptosis if they are lethal to the cell. p53, Bcl-2 and Bax play important roles in the regulation of genotoxic chemical induced cell apoptosis. Since the mechanisms by which cellular DNA damaged by different DNA-damaging chemicals may not be the same, we studied the involvement of p53, Bcl-2 and Bax in apoptosis induced by methyl methanesulfonate (MMS) and hydrogen peroxide (H2O2). H2O2 damages DNA by free radical generation and MMS damages DNA by DNA methylation. At non-lethal doses, both H2O2 and MMS induced high level of p53 protein accumulation. Nevertheless, while the amount of p53 protein increased with the dose of MMS and the occurrence of apoptotic cell death events, H2O2 doses that induce cell apoptosis attenuated the p53 protein accumulation level. Lethal MMS treatment also increased Bax, but not Bcl-2 expression, whereas in H2O2 induced apoptosis, the level of both Bcl-2 and Bax declined. These results indicate that toxic chemicals differentially regulate the accumulation of p53 protein. Thus, the pathways of toxic chemicals induced cell apoptosis are different and independent.

Transmission of de novo mutations of the deleted in azoospermia genes from a severely oligozoospermic male to a son via intracytoplasmic sperm injection.

OBJECTIVE: To investigate the transmission of microdeletions in the deleted in azoospermia (DAZ) genes to a male offspring via intracytoplasmic sperm injection (ICSI). DESIGN: Case report. SETTING: Reproductive unit of a university teaching hospital. PATIENT(S): A 29-year-old, severely oligozoospermic male with microdeletions of the DAZ genes in Yq interval 6 and his son, who was conceived via ICSI. INTERVENTION(S): DNA screening for the microdeletions in Yq interval 6 with 24 sequence tagged sites with the use of polymerase chain reaction amplification for the patient, the patient's father, and the patient's son. Paternity identification was performed using nine hypervariable short tandem repeats. MAIN OUTCOME MEASURE(S): Deletion mapping of Yq interval 6 from sequence tagged sites and electropherogram of short tandem repeats for DNA fingerprinting. RESULT(S): The son had the same microdeletions of the DAZ genes as the patient, and the patient's father had normal DAZ genes. The paternity of the patient, the patient's father, and the patient's son was verified. CONCLUSION(S): De novo DAZ microdeletions in an infertile male can be transmitted to a male offspring via ICSI. DNA screening tests for DAZ genes before ICSI may help in the genetic counseling of patients with idiopathic azoospermia or severe oligozoospermia.

Cellular properties of lateral spinal nucleus neurons in the rat L6-S1 spinal cord.

Conventional intracellular recordings were made from 26 lateral spinal nucleus (LSN) neurons in slices of L6-S1 spinal cord from 10- to 15-day-old rats. At rest, LSN neurons did not fire spontaneous action potentials. With injection of a positive current pulse, action potentials had an amplitude of 72 +/- 7 (SD) mV and duration at half-peak height of 0.75 +/- 0.22 ms. Action potentials were followed by an afterpotential. Most LSN neurons (13/17) exhibited only an afterhyperpolarization (AHP); four neurons exhibited both a fast and a slow AHP separated by an afterdepolarization (ADP). For LSN neurons that exhibited only an AHP, a slow ADP could be identified during bath application of apamin (100 nM). Four of 11 LSN neurons showed a postinhibitory rebound (PIR). Two types of PIR were noted, one with high threshold and low amplitude and the other with low threshold and high amplitude. The PIR with high amplitude was partially blocked in 0 mM Ca2+/high Mg2+ (10 mM) recording solution. Repetitive firing properties were examined in 17 LSN neurons. On the basis of the ratio of the slopes between initial instantaneous firing and steady-state firing frequencies, neurons with low spike frequency adaptation (SFA, 8/17) and high SFA (4/17) were identified. In addition, 2/17 LSN neurons exhibited biphasic repetitive firing patterns, which were composed of a fast SFA, delayed excitation, and low SFA; another two neurons showed only delayed excitation. Plateau potentials also were found in two LSN neurons. Dorsal root stimulation revealed that most LSN neurons (12/13) had polysynaptic postsynaptic potentials (PSP); only one neuron exhibited a monosynaptic PSP. Electrical stimulation of the dorsal root evoked prolonged discharges in low SFA neurons and a short discharge in high SFA neurons. Intrinsic properties were modulated by bath application of substance P (SP). Membrane potentials were depolarized in all eight LSN neurons tested, and membrane resistance was either increased (n = 3) or decreased (n = 2). Both instantaneous firing and steady-state firing were facilitated by SP. In addition, oscillation of membrane potentials were induced in three LSN neurons. These results demonstrate that LSN neurons exhibit a variety of intrinsic properties, which may significantly contribute to sensory processing, including nociceptive processing.

Association of endometrial blood flow as determined by a modified colour Doppler technique with subsequent outcome of in-vitro fertilization.

An endometrial thickness of 10 mm or more has been reported to be favourable for embryo implantation. Nevertheless, many women participating in in-vitro fertilization (IVF) programmes have adequate endometrial thickness but do not achieve satisfactory implantation. With the aid of power Doppler sonography, we examined the association between intra-endometrial vascularity and reproductive outcome. For this study, we enrolled only women with endometrial thickness >/=ISOdia>/=10 mm and excluded those with apparent endometrial pathologies. Of 95 women undergoing IVF cycles, there resulted 37 intrauterine pregnancies. The women were of similar age, body mass index, peak oestradiol concentration and endometrial thickness, and a similar number of embryos were transferred. Those women with an intra-endometrial power Doppler area (EPDA) <5 mm2 achieved a significantly lower pregnancy rate (23. 5 versus 47.5%, P = 0.021) and implantation rate (8.1 versus 20.2%, P = 0.003) than those with an EPDA >/=ISOdia>/=5 mm2. We conclude that, in addition to endometrial thickness, EPDA may serve as a factor indicative of endometrial receptivity. Women with adequate endometrial thickness but a small EPDA tended to have an unfavourable reproductive outcome.

Participation of central descending nociceptive facilitatory systems in secondary hyperalgesia produced by mustard oil.

The present series of experiments were designed to examine a potential role for central descending pain facilitatory systems in mediating secondary hyperalgesia produced by topical application of mustard oil and measuring the nociceptive tail-flick reflex in awake rats. Topical application of mustard oil (100%) to the lateral surface of the hind leg produced a facilitation of the tail-flick reflex that was significantly reduced in spinal transected animals. Mustard oil hyperalgesia was also inhibited in animals that had received electrolytic lesions in the rostral ventromedial medulla (RVM). Intrathecal (i.t.) administration of the non-selective cholecystokinin (CCK) receptor antagonist proglumide (10 micrograms) prior to mustard oil application completely blocked both the lesser and greater hyperalgesic responses observed in spinal transected and normal animals, respectively, and produced an inhibition of the tail-flick reflex in normal animals. Administration of the selective CCKB receptor antagonist L-365260 i.t. dose-dependently inhibited mustard oil hyperalgesia (ID50 = 364 ng) at doses approximately 5-fold less than the CCKA receptor antagonist devazepide (ID50 = 1760 ng). Similar to spinal proglumide, microinjection of the neurotensin antagonist SR48692 (3.5 micrograms) into the RVM blocked mustard oil hyperalgesia and inhibited the tail-flick reflex. These data suggest that secondary hyperalgesia produced by mustard oil is mediated largely by a central, centrifugal descending pain facilitatory system which involves neurotensin in the RVM and spinal CCK (via CCKB receptors). The inhibition of the tail-flick reflex produced by mustard oil following spinal or supraspinal administration of receptor antagonists suggests concurrent activation of central descending facilitatory and inhibitory systems.

Inhibition of HIV-1 Tat-mediated transactivation by quinacrine and chloroquine.

The replication of human immunodeficiency virus type 1 (HIV-1) requires cellular components to interact with regulatory elements located in the long terminal repeat (LTR) as well as viral proteins Tat and Rev. Several well known signaling transduction inhibitors were tested to determine their effects on the Tat-mediated transactivation using a transfection assay with the bacterial chloramphenicol acetyltransferase gene under the control of the HIV-1 LTR. The protein kinase C inhibitors curcumin and staurosporine, but not a tyrosine kinase inhibitor herbimycine A, inhibited Tat-mediated LTR-driven transactivation. Two antimalarial drugs quinacrine and chloroquine, that are also arachidonic acid metabolism inhibitors, were found to inhibit the Tat-mediated LTR-driven gene expression. Another inhibitor of arachidonic acid metabolism 4-bromophenacyl bromide was also found to inhibit Tat-mediated gene expression driven by HIV-1 LTR. However, the antimalarial drug quinine elicited no effects on Tat-mediated transactivation. These results suggest that the anti-arachidonic acid metabolism properties of quinacrine and chloroquine may be responsible for their ability to inhibit Tat-mediated LTR-regulated gene expression.

Differential regulation of p53, c-Myc, Bcl-2 and Bax protein expression during apoptosis induced by widely divergent stimuli in human hepatoblastoma cells.

Apoptosis of HepG2 cells triggered by various agents is characterized in an attempt to delineate the common apoptosis signaling pathway in human hepatoma cells. Several hallmarks of apoptosis, including DNA laddering, chromatin condensation and fragmentation, and an apoptosis specific cleavage of 28S and 18S ribosomal RNA were observed after treatment with curcumin. Curcumin treatment however did not alter the expression levels of Bcl-2 and Bax proteins. p53 protein accumulated slowly and decreased abruptly after reaching the maximum. Conversely, c-Myc protein decreased initially and subsequently increased preceding the onset of apoptosis. The accumulation of p53 protein is not due to increased levels of p53 mRNA and does not result in growth arrest. Staurosporine, quinacrine, ultraviolet irradiation, hydrogen peroxide, and cyclohexamide are all capable of triggering apoptosis in HepG2 cells. While most of these agents affect the expression levels of p53 and c-Myc similarly, none of them altered the expression levels of the Bcl-2 and Bax proteins. In conclusion, these data suggest that p53 and c-Myc may play a more important role in the apoptosis signaling pathway in HepG2 cells, than the bcl-2 gene family.

Curcumin induces apoptosis in immortalized NIH 3T3 and malignant cancer cell lines.

Curcumin, which is a widely used dietary pigment and spice, has been demonstrated to be an effective inhibitor of tumor promotion in mouse skin carcinogenesis. We report that curcumin induces cell shrinkage, chromatin condensation, and DNA fragmentation, characteristics of apoptosis, in immortalized mouse embryo fibroblast NIH 3T3 erb B2 oncogene-transformed NIH 3T3, mouse sarcoma S180, human colon cancer cell HT-29, human kidney cancer cell 293, and human hepatocellular carcinoma Hep G2 cells, but not in primary culture of mouse embryonic fibroblast C3H 10T1/2, rat embryonic fibroblast, and human foreskin fibroblast cells in a concentration- and time-dependent manner. Many cellular and biochemical effects of curcumin in mouse fibroblast cells have been reported, such as inhibition of protein kinase C (PKC) activity induced by phorbol 12-myristate 13-acetate treatment, inhibition of tyrosine protein kinase activity, and inhibition of arachidonic acid (AA) metabolism. Treatment of NIH 3T3 cells with the PKC inhibitor staurosporine, the tyrosine kinase inhibitor herbimycin A, and the AA metabolism inhibitor quinacrine induces apoptotic cell death. These results suggest that, in some immortalized and transformed cells, blocking the cellular signal transduction might trigger the induction of apoptosis.