Comparison between Dual-Energy CT and Quantitative Susceptibility Mapping in Assessing Brain Iron Deposition in Parkinson Disease.

BACKGROUND AND PURPOSE: Both dual-energy CT and quantitative susceptibility mapping can evaluate iron depositions in the brain. The purpose of this study was to compare these 2 techniques in evaluating brain iron depositions in Parkinson disease. MATERIALS AND METHODS: Forty-one patients with Parkinson disease (Parkinson disease group) and 31 age- and sex-matched healthy controls (healthy control group) were included. All participants underwent brain dual-energy CT and quantitative susceptibility mapping. ROIs were set bilaterally in the globus pallidus, substantia nigra, red nucleus, caudate nucleus, and putamen. CT values and magnetic susceptibility values were obtained in each ROI. Differences in CT values and magnetic susceptibility values between the Parkinson disease and healthy control groups were compared, followed by analysis of receiver operating characteristic curves. Correlations between CT values and magnetic susceptibility values were then evaluated. RESULTS: The CT values of the bilateral globus pallidus, substantia nigra, and red nucleus were higher in the Parkinson disease group (P < .05). The magnetic susceptibility values of the bilateral globus pallidus and substantia nigra were higher in the Parkinson disease group (P < .05). The CT value of the right globus pallidus in linear fusion images had the highest diagnostic performance (0.912). Magnetic susceptibility values of the bilateral globus pallidus in the Parkinson disease group were positively correlated with CT values at the level of 80 kV(peak), linear fusion images, and SN150 kV(p) (r = 0.466∼0.617; all, P < .05). CONCLUSIONS: Both dual-energy CT and quantitative susceptibility mapping could assess excessive brain iron depositions in Parkinson disease, and we found a positive correlation between CT values and magnetic susceptibility values in the bilateral globus pallidus.

[Recurrence and influencing factors of diabetic foot ulcer in patients with type 2 diabetes mellitus].

Objective: To investigate the recurrence and influencing factors of diabetic foot ulcer in patients with type 2 diabetes mellitus. Methods: Totally 185 type 2 diabetes patients with new-onset of diabetic foot ulcers admitted to Fuyang People's Hospital of Anhui Province from January 2011 to December 2015 were enrolled in this study, including 120 males and 65 females, aged 40-79 years. All the patients were followed up for 3 years, and their clinical data were retrospectively analyzed by the case-control study. The Kaplan-Meier cumulative recurrence curve was drawn according to the 3-year cumulative recurrence rate of diabetic foot ulcers. The time to visit, toe involvement, and amputation of involved toes in patients with recurrent diabetic foot ulcer were counted at the initial onset and the recurrence of the ulcers, respectively, and the data were statistically analyzed with t test and chi-square test. According to the recurrence of diabetic foot ulcers, the patients were divided into foot ulcer recurrence group and foot ulcer non-recurrence group. The gender, age, course of diabetes mellitus, length of hospital stay, visit time, body mass index, glycosylated hemoglobin HbA1c, total bilirubin, albumin, creatinine, cholesterol, low density lipoprotein (LDL), high density lipoprotein (HDL), triglycerides, hemoglobin, white blood cell count, toe involvement, toe amputation, ankle-brachial index, diabetic retinopathy (DR), diabetic peripheral neuropathy (DPN), diabetic nephropathy (DN), history of hypertension, cardio-cerebrovascular disease, smoking, residence, solitary life, and walking disorder of patients between the two groups were compared, and the data were statistically analyzed with t test and chi-square test. Log-rank test was performed on the indexes with P<0.1 in comparison between two groups, and the indexes with statistically significant differences in Log-rank test were analyzed by multivariate Cox regression analysis to screen the influencing factors of recurrence of diabetic foot ulcer. Results: (1) The 3-year cumulative recurrence rate of diabetic foot ulcers in 185 patients with type 2 diabetes mellitus was 47.0% (87/185). (2) For 87 patients with diabetic foot ulcer recurrence, compared with that at the initial onset of the ulcers, the visit time was significantly shorter (t=10.593, P<0.01), the toe amputation rate was significantly increased (χ(2)=5.118, P<0.05), but there was no obvious change in toe involvement at the recurrence of the ulcers. (3) There were statistically significant differences in age, course of diabetes mellitus, length of hospital stay, body mass index, glycosylated hemoglobin HbA1c, total bilirubin, albumin, creatinine, cholesterol, LDL, HDL, hemoglobin, white blood cell count, gender, toe amputation, ankle-brachial index, DR, history of cardio-cerebrovascular disease, solitary life, and walking disorder of patients between foot ulcer recurrence group (87 patients) and foot ulcer non-recurrence group (98 patients) (t=5.123, 4.242, 5.324, -24.572, 6.102, -1.984, -9.747, 3.226, 3.076, 3.646, -4.683, -7.502, 8.095, χ(2)=5.621, 18.433, 4.546, 5.785, 9.655, 7.625, 7.886, P<0.05 or P<0.01), while the rest of the indexes of patients between the two groups were similar. Log-rank test showed that the two groups had statistically significant differences in age, course of diabetes mellitus, length of hospital stay, glycosylated hemoglobin HbA1c, total bilirubin, albumin, creatinine, ankle-brachial index, DPN, and walking disorder (χ(2)=210.046, 44.837, 34.107, 98.685, 66.532, 294.451, 260.554, 5.012, 6.818, 11.160, P<0.05 or P<0.01). Age, total bilirubin, albumin, DPN, and walking disorder were the influencing factors for the recurrence of diabetic foot ulcers in patients with type 2 diabetes mellitus (hazard ratio=1.024, 0.678, 0.849, 2.335, 4.099, 95% confidence interval=1.001-1.047, 0.558-0.823, 0.797-0.904, 1.280-4.258, 2.044-8.223, P<0.05 or P<0.01). Conclusions: The 3-year cumulative recurrence rate of diabetic foot ulcers in patients with type 2 diabetes mellitus is relatively high, with the influencing factors being age, total bilirubin, albumin, DPN, and walking disorder.

Clinical implications of head-shaking nystagmus in central and peripheral vestibular disorders: is perverted head-shaking nystagmus specific for central vestibular pathology?

BACKGROUND AND PURPOSE: The patterns of head-shaking nystagmus (HSN) aid in differentiation between central and peripheral vestibular disorders, and perverted HSN (pHSN) has been considered a central sign. The aim was to determine the characteristics of HSN in a large number of patients with either peripheral or central vestibular disorders in a dizziness clinic of a university hospital. METHODS: The medical records of 7544 dizzy patients were reviewed during a year and 822 patients with a clinical diagnosis of vestibular disorders were recruited. The findings of spontaneous nystagmus (SN) and HSN in these patients were compared with those of healthy controls (n = 48). RESULTS: A total of 217 of the 822 patients (26.4%) were classified as having a central vestibular disorder, whilst 397 (48.3%) had a peripheral vestibular disorder. In the peripheral vestibular disorder group, SN was observed in 14.1% and HSN in 40.8%, amongst whom 24.1% were the pHSN form. In the central group, SN was observed in 17.5% and HSN in 24.0% of whom 57.7% was pHSN. HSN was more frequently observed in the peripheral vestibular disorder group than in the central group (40.8% vs. 24.0%, P < 0.01). However, the proportion of pHSN was significantly increased in the central group compared to the peripheral vestibular patient group (57.7% vs. 24.1%, P < 0.01). CONCLUSIONS: Since pHSN is not specific for central vestibular disorders, other clinical features should be considered in pursuing a central lesion in patients with pHSN.

Antimicrobial activity of recombinant mature bovine neutrophil ß-defensin 4 on mycobacterial infection.

BACKGROUND: Previous studies have shown that human cathelicidin and defensins have effective antimicrobial activity against Mycobacterium spp. OBJECTIVE: To investigate the antimycobacterial effect of mature bovine neutrophil ß-defensin (mBNBD) 4 against Mycobacterium spp. infection for the first time. DESIGN: mBNBD4 protein was expressed in Pichia pastoris GS115. We used immunofluorescent assay to detect whether the recombinant mBNBD4 had entered the macrophages. The antimycobacterial activity of mBNBD4 was tested through colony-forming unit (cfu) assay. Morphological changes in the cell wall of M. bovis treated with mBNBD4 were observed by scanning electron microscope. RESULTS: mBNBD4 was expressed and successfully purified from P. pastoris with intact antimicrobial activity. The recombinant protein was able to enter Raw 264.7 macrophages and exhibited potent in vitro bactericidal activity against M. smegmatis and M. bovis. The cell wall of M. bovis was disrupted after interaction with mBNBD4. Exogenous addition of mBNBD4 to both Raw 264.7 and THP-1 derived macrophages reduced the intracellular survival of M. bovis and M. tuberculosis relative to control cells. CONCLUSION: Our data show that mBNBD4 plays an important role in inhibiting mycobacterial growth and in controlling intracellular survival of mycobacteria. mBNBD4 could therefore an effective antimycobacterial molecule in combination with other measures.

Cell cycle and immune-related processes are significantly altered in chronic GVHD.

Currently, the pathogenesis of chronic GVHD is unclear. To elucidate the molecular characteristics underlying chronic GVHD, we analyzed the gene expression profiles of 21 mononuclear cell samples from allogeneic hematopoietic stem cell transplantation (HSCT) recipients. Self organizing map (SOM) clustering showed that the entire expression profiles of chronic GVHD samples were clearly different from those of the non-GVHD samples, and significance analysis of microarray (SAM) demonstrated that 120 genes, including PTDSS1, VAV1 and CD3D, were up-regulated, and 5 genes, including calnexin, were down-regulated in GVHD patients. Gene ontology annotation revealed that these genes are related to the phosphorous metabolism and lipid biosynthesis. Quantitative real time polymerase chain reaction (qRT-PCR) experiments validated the up-regulation of PTDSS1, VAV1 and CD3D in separate samples. Pathway-wise global test revealed that differential gene expression in cell cycle and T cell immune-associated pathways were significant between GVHD patients and non-GVHD patients. Seventeen classifier genes selected using a PAM (prediction analysis of microarray) algorithm showed favorable performance (prediction accuracy=0.85) for identifying patients with chronic GVHD. In conclusion, we identified differentially expressed genes and pathways in chronic GVHD patients using microarray analysis, and we also selected diagnostic genes predicting chronic GVHD status.

Advances in renal (patho)physiology using multiphoton microscopy.

Multiphoton excitation fluorescence microscopy is a state-of-the-art confocal imaging technique ideal for deep optical sectioning of living tissues. It is capable of performing ultrasensitive, quantitative imaging of organ functions in health and disease with high spatial and temporal resolution which other imaging modalities cannot achieve. For more than a decade, multiphoton microscopy has been successfully used with various in vitro and in vivo experimental approaches to study many functions of different organs, including the kidney. This study focuses on recent advances in our knowledge of renal (patho)physiological processes made possible by the use of this imaging technology. Visualization of cellular variables like cytosolic calcium, pH, cell-to-cell communication and signal propagation, interstitial fluid flow in the juxtaglomerular apparatus (JGA), real-time imaging of tubuloglomerular feedback (TGF), and renin release mechanisms are reviewed. A brief summary is provided of kidney functions that can be measured by in vivo quantitative multiphoton imaging including glomerular filtration and permeability, concentration, dilution, and activity of the intrarenal renin-angiotensin system using this minimally invasive approach. New visual data challenge a number of existing paradigms in renal (patho)physiology. Also, quantitative imaging of kidney function with multiphoton microscopy has tremendous potential to eventually provide novel non-invasive diagnostic and therapeutic tools for future applications in clinical nephrology.

Cholesterol-3-beta, 5-alpha, 6-beta-triol induced genotoxicity through reactive oxygen species formation.

The mutagenicity of oxysterols, cholesterol-3beta,5alpha,6beta-triol (alpha-Triol), 7-keto-cholesterol (7-Keto) and cholesterol-5alpha,6alpha-epoxide (alpha-Epox) were examined by the Ames method and chromosome aberration test in this study. Only alpha-Triol concentration-dependently caused an increase of bacterial revertants in the absence of metabolic activating enzymes (S9), but not 7-keto and alpha-Epox. The mutagenic effect of alpha-Triol was reduced by the addition of S9. On the other hand, although alpha-Triol significantly induced chromosome aberration in CHO-K1 cells with and without S9. However, the addition of S9 reduced the degree of abnormal structure chromosome compared to without S9 mix. Catalase and superoxide dismutase (SOD) inhibited alpha-Triol induced increase of revertants in Salmonella typhimurium and chromosome aberration frequency in CHO cells, suggesting that reactive oxygen species (ROS) might be involved in the genotoxic effect of alpha-Triol. Treatment with alpha-Triol increased the ROS production in CHO cells, which could be attenuated by catalase and SOD. Results in this study suggested, for the first time that alpha-Triol, causes genotoxic effect in an ROS-dependent manner.

Reactive oxygen species are crucial for hydroxychavicol toxicity toward KB epithelial cells.

Betel quid (BQ) chewing shows a strong correlation to the incidence of oral submucous fibrosis (OSF), leukoplakia and oral cancer. BQ contains mainly areca nut, lime, Piper betle leaf (PBL) and the inflorescence of P. betle (IPB). Hydroxychavicol (4-allyl-catechol, HC), as a major phenolic compound in PBL and IPB, is shown to induce oxidative stress, glutathione (GSH) depletion and cell cycle deregulation. Using bivariate BrdU/PI flow cytometry, KB cells in DNA synthesis (S phase) are shown to be sensitive to the toxic effect of HC and show cell cycle arrest and apoptosis following exposure to 0.1 and 0.3 mM HC. HC-induced apoptosis and cell cycle arrest are associated with mitochondrial membrane potential (delta Psim) depolarization as revealed by a decrease in rhodamine fluorescence. N-acetyl-L-cysteine (1 mM), superoxide dismutase (100 U/ml) and catalase (1000 U/ml) were effective in prevention of HC-induced GSH depletion (as indicated by chloromethylfluorescein fluorescence), reactive oxygen species (ROS) production (by dichlorofluorescein fluorescence), cell cycle arrest and apoptosis. However, dimethylthiourea (2 mM), neocuproine (1 mM), 1,10-phenanthroline (200 microM) and desferrioxamine (0.5 mM) showed little effect on HC-induced cell changes. HC elevated the cellular and mitochondrial GSH levels at moderate concentrations (0.05-0.1 mM), whereas at a concentration of 0.3 mM, inhibitory effects were noted. These results indicate that HC consumption may be associated with BQ-chewing-related oral mucosal diseases via GSH depletion, ROS production, mitochondrial dysfunction, cell cycle disturbance and the induction of apoptosis. These events are related to the production of superoxide radicals and hydrogen peroxide.

Quantitation of Na+-K+-2Cl- cotransport splice variants in human tissues using kinetic polymerase chain reaction.

A kinetic reverse transcription-polymerase chain reaction (RT-PCR)-based assay is described that can discriminate and quantitate differentially spliced mRNAs. This assay should be generally applicable for high-throughput quantitation of differentially spliced transcripts. The utility of this method was assessed for spliced transcripts encoded by the human Na+-K+-2Cl- cotransporter gene hNKCC1. Evidence is presented that the NKCC1 isoform of the human Na+-K+-2Cl- cotransporter is differentially spliced analogous to that recently described for the mouse Na+-K+-2Cl- cotransporter gene BSC2. The nucleotide sequences of the two human splice variants predict Na+-K+-2Cl- cotransporter proteins differing only in length. Stable transfectants expressing these human splice variants, designated NKCC1a or NKCC1b, were constructed. Both splice variants produce functional Na+-K+-2Cl- cotransporters in vivo. The abundance of NKCC1 mRNA and patterns of differential splicing in 10 different tissue types and three cell lines were quantitated using the kRT-PCR assay. The results showed that the total amount of NKCC1 mRNA varied by more than 30-fold in the human tissues and cell lines examined. The ratio of NKCC1a/NKCC1b varied nearly 70-fold among these same tissues and cell lines suggesting that differential splicing of the NKCC1 transcript may play a regulatory role in human tissues.

Triptolide and chemotherapy cooperate in tumor cell apoptosis. A role for the p53 pathway.

Triptolide (PG490), a diterpene triepoxide, is a potent immunosuppressive agent extracted from the Chinese herb Tripterygium wilfordii. We have previously shown that triptolide blocks NF-kappaB activation and sensitizes tumor necrosis factor (TNF-alpha)-resistant tumor cell lines to TNF-alpha-induced apoptosis. We show here that triptolide enhances chemotherapy-induced apoptosis. In triptolide-treated cells, the expression of p53 increased but the transcriptional function of p53 was inhibited, and we observed a down-regulation of p21(waf1/cip1), a p53-responsive gene. The increase in levels of the p53 protein was mediated by enhanced translation of the p53 protein. Additionally, triptolide induced accumulation of cells in S phase and blocked doxorubicin-mediated accumulation of cells in G(2)/M and doxorubicin-mediated induction of p21. Our data suggest that triptolide, by blocking p21-mediated growth arrest, enhances apoptosis in tumor cells.

Synthesis, antiplatelet and vasorelaxing effects of monooxygenated flavones and flavonoxypropanolamines.

A series of flavones and flavonoxypropanolamines were synthesized and tested in-vitro for their ability to inhibit aggregation of washed rabbit platelets and human platelet-rich plasma (PRP), and for vasoconstriction of rat thoracic aorta. The various substituted positions of the hydroxyl group in flavone ring B and the various oxypropanolamine side chains substituted at position C-2' of flavone modified the antiplatelet effects. All the compounds tested in human PRP showed significant inhibition of secondary aggregation induced by adrenaline (epinephrine), suggesting that the antiplatelet effect of these compounds is mainly due to an inhibitory effect on thromboxane formation. Compounds 11 and 12 also had potent vasorelaxant effects in rat thoracic aorta. Phenylephrine- and high-K+-induced 45Ca2+ influx in aorta were both inhibited by the selected compound 11. This result indicates that the inhibitory effect of 11 on the contractile response caused by high-K+ medium and noradrenaline (norepinephrine) in rat thoracic aorta is mainly due to inhibition of Ca2+ influx through both voltage-dependent and receptor-operated Ca2+ channels.

Effects of cartap on isolated mouse phrenic nerve diaphragm and its related mechanism.

Cartap, a nereistoxin analogue pesticide, is reported to have no irritation to eyes in rabbits. However, we have demonstrated recently that cartap could actually cause acute death in rabbits via ocular exposure. Our preliminary study with isolated mouse phrenic nerve diaphragms has shown that instead of neuromuscular blockade, cartap caused muscular contracture. The objective of the study was to examine the effect of cartap on the neuromuscular junction in more detail and to investigate its possible underlying mechanism with isolated mouse phrenic nerve diaphragms and sarcoplasmic reticulum (SR) vesicles. Cartap or nereistoxin at various concentrations was added in the organ bath with isolated mouse phrenic nerve diaphragm and both nerve- and muscle-evoked twitches were recorded. Instead of blocking the neuromuscular transmission as nereistoxin did, cartap caused contracture in stimulated or quiescent isolated mouse phrenic nerve diaphragm. Both the cartap-induced muscular contracture force and the time interval to initiate the contracture were dose-dependent. The contracture induced by cartap was not affected by the pretreatment of the diaphragm with the acetylcholine receptor blocker alpha-bungarotoxin; the Na(+) channel blocker tetrodotoxin; or various Ca(2+) channel blockers, NiCl(2), verapamil, and nifedipine. On the contrary, the contracture was significantly inhibited when the diaphragm was pretreated with ryanodine or EGTA containing Ca(2+)-free Krebs solution or in combination. This suggested that both internal and extracellular Ca(2+) might participate in cartap-induced skeletal muscle contracture. Moreover, cartap inhibited the [(3)H]-ryanodine binding to the Ca(2+) release channel of SR in a dose-dependent manner. Additionally, cartap could induce a significant reduction in Ca(2+)-ATPase activity of SR vesicles at a relatively high dose. The results suggested that cartap might cause the influx of extracellular Ca(2+) and the release of internal Ca(2+), with subsequent induction of muscular contracture in the isolated mouse phrenic nerve diaphragm. Based on these findings, we propose that the acute death of rabbits following ocular exposure to cartap might have resulted from respiratory failure secondary to diaphragm contracture.

Induction of skeletal muscle contracture and calcium release from isolated sarcoplasmic reticulum vesicles by sanguinarine.

The benzophenanthrine alkaloid, sanguinarine, was studied for its effects on isolated mouse phrenic-nerve diaphragm preparations. Sanguinarine induced direct, dose-dependent effects on muscle contractility. Sanguinarine-induced contracture was partially inhibited when the extracellular Ca(2+) was removed or when the diaphragm was pretreated with nifedipine. Depletion of sarcoplasmic reticulum (SR) internal calcium stores completely blocked the contracture. Sanguinarine induced Ca(2+) release from the actively loaded SR vesicles was blocked by ruthenium red and dithiothreitol (DTT), consistent with the ryanodine receptor (RyR) as the site of sanguinarine action. Sanguinarine altered [(3)H]-ryanodine binding to the RyR of isolated SR vesicles, potentiating [(3)H]-ryanodine binding at lower concentrations and inhibiting binding at higher concentrations. All of these effects were reversed by DTT, suggesting that sanguinarine-induced Ca(2+) release from SR occurs through oxidation of critical SH groups of the RyR SR calcium release channel.

Alterations in the properties and isoforms of sciatic nerve Na(+), K(+)-ATPase in methylcyclopentadienyl manganese tricarbonyl-treated mice.

The in vivo effect of methylcyclopentadienyl manganese tricarbonyl (MMT), an organic manganese-containing compound, on the mouse motor nerve was studied. The motor nerve conduction velocity was markedly decreased in MMT-treated mice. The Na(+),K(+)-ATPase activity of sciatic nerve isolated from MMT-treated mice was decreased; however, the sciatic nerve Na(+),K(+)-ATPase activity was not affected by the in vitro treatment of MMT. Moreover, [(3)H]ouabain binding of sciatic nerve isolated from MMT-treated mice was decreased. Using Western blot analysis, the amount of Na(+),K(+)-ATPase catalytic alpha1 subunit polypeptide in sciatic nerve of MMT-treated mice was also decreased. These results indicate that a causal relationship may exist between reduced nerve Na(+),K(+)-ATPase activity and motor nerve conduction velocity in MMT-treated mice and that a measurable decrease in alpha1 catalytic subunit isoform of Na(+),K(+)-ATPase may be necessary for the development of peripheral neuropathy by MMT.

Transcript quantitation in total yeast cellular RNA using kinetic PCR.

Kinetically monitored, reverse transcriptase-initiated PCR (kinetic RT-PCR, kRT-PCR) is a novel application of kinetic PCR for high throughput transcript quantitation in total cellular RNA. The assay offers the simplicity and flexibility of an enzyme assay with distinct advantages over DNA microarray hybridization and SAGE technologies for certain applications. The reproducibility, sensitivity and accuracy of the kRT-PCR were assessed for yeast transcripts previously quantitated by a variety of methods including SAGE analysis. Changes in transcript levels between different genetic or physiological cell states were reproducibly quantitated with an accuracy of +/-20%. The assay was sufficiently sensitive to quantitate yeast transcripts over a range of more than five orders of magnitude, including low abundance transcripts encoding cell cycle and transcriptional regulators.

Inhibition of agonist-induced vasocontraction and impairment of endothelium-dependent vasorelaxation by extract of motorcycle exhaust particles in vitro.

The in vitro effects of motorcycle exhaust particulate extract (MEPE) on blood vessels were studied in thoracic aorta isolated from Wistar rat. The MEPE relaxed the phenylephrine-precontracted aorta with an EC50 value of 0.05 +/- 0.004 mg/ml. This relaxing effect of MEPE persisted in endothelium-denuded aorta, suggesting that the relaxation induced by MEPE is endothelium-independent. The phenylephrine-induced vasocontraction and inositol 1,4,5-triphosphate formation were inhibited concentration dependently in aorta pretreated with MEPE. However, the high-K+-induced vasocontraction and the Ca2+ sensitivity of the contractile proteins were not significantly affected by MEPE. In addition to the inhibitory effects on agonist-induced contraction, the vasorelaxing effects both of acetylcholine and of sodium nitroprusside were impaired by MEPE. The inhibitory effects of MEPE on acetylcholine and sodium nitroprusside, but not phenylephrine, were reversed by cotreatment with superoxide dismutase. These results showed that the MEPE, added in vitro, inhibited the phenylephrine-induced, but not depolarization-induced, vasocontraction of aorta. The MEPE also impaired the vasorelaxation induced by acetylcholine in a superoxide anion-dependent manner.

Cascades of mammalian caspase activation in the yeast Saccharomyces cerevisiae.

Caspases (aspartate-specific cysteine proteases) play a critical role in the execution of the mammalian apoptotic program. To address the regulation of human caspase activation, we used the yeast Saccharomyces cerevisiae, which is devoid of endogenous caspases. The apical procaspases, -8beta and -10, were efficiently processed and activated in yeast. Although protease activity, per se, was insufficient to drive cell death, caspase-10 activity had little effect on cell viability, whereas expression of caspase-8beta was cytotoxic. This lethal phenotype was abrogated by co-expression of the pan-caspase inhibitor, baculovirus p35, and by mutation of the active site cysteine of procaspase-8beta. In contrast, autoactivation of the executioner caspase-3 and -6 zymogens was not detected. Procaspase-3 activation required co-expression of procaspase-8 or -10. Surprisingly, activation of procaspase-6 required proteolytic activities other than caspase-8, -10, or -3. Caspase-8beta or -10 activity was insufficient to catalyze the maturation of procaspase-6. Moreover, a constitutively active caspase-3, although cytotoxic in its own right, was unable to induce the processing of wild-type procaspase-6 and vice versa. These results distinguish sequential modes of activation for different caspases in vivo and establish a yeast model system to examine the regulation of caspase cascades. Moreover, the distinct terminal phenotypes induced by various caspases attest to differences in the cellular targets of these apoptotic proteases, which may be defined using this system.

Mediation of gamma-hexachlorocyclohexane-induced DNA fragmentation in HL-60 cells through intracellular Ca2+ release pathway.

The cytotoxicity of gamma-hexachlorcyclohexane (gamma-HCC) was evaluated in HL-60 cells. Gamma-HCC dose-dependently induced cytotoxicity of HL-60 with an IC50 value of 60+/-5 microM. The gamma-HCC treated cells showed some characteristic changes of apoptosis, including blebbing of the membrane, condensation of the nuclear chromatin, vacuolation of cytoplasm and internucleosomal DNA fragmentation. Gamma-HCC induced DNA fragmentation of HL-60 cells in a dose-, time- and Ca2+-dependent manner. The DNA fragmentation induced was inhibited by intracellular Ca2+ chelator, calmodulin antagonist and Ca2+ sensitive endonuclease inhibitor. Gamma-HCC caused a steady increase in the cytosolic free Ca+ concentration due to release from intracellular stores. Neither the DNA fragmentation nor the increase of intracellular Ca2+ induced by gamma-HCC was inhibited by the removal of extracellular Ca2+. These data suggested that the cytotoxicity of gamma-HCC in HL-60 cells is mediated by the increase of intracellular Ca2+ concentration and the activation of Ca2+-dependent endonuclease, which triggers apoptosis in a Ca2+ and calmodulin-dependent manner.

Analysis of colorectal cancer by comparative genomic hybridization: evidence for induction of the metastatic phenotype by loss of tumor suppressor genes.

Current models suggest that colon cancer initiation and progression are secondary to both the activation of oncogenes and the deletion of tumor suppressor genes. The role of each, however, is still poorly understood, particularly with regard to the induction of metastasis. We hypothesized that genetic differences exist between tumors that metastasize distantly and those that do not, and that oncogenes and tumor suppressor genes participate equally in this process. To address this hypothesis, human tumor specimens from localized [tumor-node-metastasis (TNM) stage I-III] and primary colon cancers (n = 10) were directly compared with metastatic (TNM stage IV) lesions (n = 10) using comparative genomic hybridization analysis. Although several alterations were shared equally between primary tumors and metastases (+7q, +19q, and +20q), two patterns of distinguishing alterations were observed: (a) alterations that were more extensive in liver metastases than in primary tumors (+8q, +13q, -4p, -8p, -15q, -17p, -18q, -21q, and -22q); and (b) alterations that were unique to metastatic lesions (-9q, -11q, and -17q). Overall, genetic losses were more common than gains, and, more importantly, the number of losses/tumor was significantly higher for metastases than for primary tumors (9.3 + 1.3 versus 4.1 + 0.7; P = 0.00062, Wilcoxon's rank-sum test). The distinct predominance of genetic losses in the metastatic lesions when compared with the primary localized tumors provides evidence that the metastatic phenotype is induced by the deletion of tumor suppressor genes and permits the construction of physical maps targeting regions where novel tumor suppressor genes are likely to exist.

Comparative studies on the induction of muscle contracture in mouse diaphragm and Ca2+ release from sarcoplasmic reticulum vesicles by organotin compounds.

Effects of organotins, including triethyltin and tributyltin, on skeletal muscle were studied with diaphragm and isolated sarcoplasmic reticulum membrane vesicles. Triethyltin induced muscle contracture in mouse diaphragm while tributyltin had comparatively less potency and efficacy in inducing the muscle contracture. The contracture induced by tributyltin was inhibited when the diaphragm was pretreated with low Ca2+ medium or caffeine while the contracture induced by triethyltin persisted in the Ca2+-free medium but was inhibited by pretreatment of caffeine. Pretreatment of dithiothreitol blocked the contracture induced by tributyltin but not that by triethyltin. Triethyltin dose-dependently induced Ca2+ release from sarcoplasmic reticulum vesicles and inhibited the Ca2+-ATPase activity. These results suggested that triethyltin induced contracture in mouse diaphragm was mainly by induction of Ca2+ release and inhibition of Ca2+ uptake of the internal Ca2+ storage site the sarcoplasmic reticulum, while the tributyltin induced contracture might be due to enhancement of extracellular Ca2+ influx which further induce the release of internal Ca2+ through the Ca2+-induced Ca2+ release mechanism.