Altered PTEN function caused by deletion or gene disruption is associated with poor prognosis in rectal but not in colon cancer.

Colorectal cancer is the third most common malignancy worldwide. Anti-epidermal growth factor receptor (EGFR)-targeted therapy shows clinical evidence in this malignancy and improves outcome. The tumor suppressor gene phosphatase and tensin homologue (PTEN) is considered a potential predictor of nonresponse to anti-EGFR agents. The purpose of this study was to assess whether associations between PTEN alterations (PTEN gene deletion or PTEN gene disruption) and clinical outcome could be caused by a prognostic (and not predictive) effect of PTEN inactivation. Therefore, we analyzed 404 colorectal cancers not previously treated with anti-EGFR drugs in a tissue microarray format. PTEN deletion and PTEN gene rearrangements were analyzed by fluorescence in situ hybridization. Heterogeneity analysis of all available large tissue sections was performed in 6 cases with genomic PTEN alteration. Twenty-seven (8.8%) of 307 analyzable colorectal cancer spots showed genomic PTEN alterations including 24 hemizygous and 1 homozygous deletion as well as 2 PTEN gene disruptions. Genomic PTEN alterations were associated with reduced patient survival in rectal cancer in univariate and multivariate analyses (P = .012; hazard ratio, 2.675; 95% confidence interval, 1.242-5.759) but not in colon cancer. Large-section evaluation revealed a homogeneous distribution pattern in all 4 analyzed cases with PTEN deletion and in both cases with a PTEN gene disruption. In conclusion, genomic PTEN gene alterations caused by deletion or gene disruption characterize a fraction of rectal cancers with particularly poor outcome.

Occurrence of Late Blight Caused by Phytophthora infestans on Potato and Tomato in Alaska.

Phytophthora infestans, causal agent of late blight, was included in a list of plant pathogens found in Alaska in 1934 (1). No notes of symptoms, extent of disease, or dates were recorded. The only reference to the location was given as Wrangell, a town in southeast Alaska with subsistence gardening. Neither P. infestans nor late blight was noted again in the state for another 59 years. Late blight first appeared in Alaska's major potato-growing region in south-central Alaska's Matanuska Valley in 1995. Subsequent outbreaks have been sporadic, occurring only in 1998, 2005, and 2006. Each of these outbreaks was identified from rapidly enlarging brown foliar lesions with branched sporangiophores and lemon-shaped sporangia (~25 × 30 µm). The 1995 and 1998 potato late blight outbreaks in Alaska were not sampled extensively nor have they previously been formally reported. We recovered single isolates of P. infestans from symptomatic potato foliage in the 1995 and 1998 outbreaks. In 2005, symptomatic foliage was collected from individual potato plants in 10 commercial fields and from tomato plants in greenhouses at two locations. Sporulating stem and leaf tissue were used to inoculate semiselective rye medium and 147 isolates from potato and six from tomato were recovered. The isolates from the 1995, 1998, and 2005 outbreaks were analyzed to determine genotype at the allozyme loci GPI and PEP (3), mitochondrial haplotype (4), mating type, and metalaxyl sensitivity (2). The 1998 and 2005 outbreaks were similar because both were caused by the relatively aggressive US-11 allozyme genotype and had significant economic impact for commercial potato growers. All 153 isolates from potato and tomato in 2005 displayed the same allozyme pattern as the US-11 genotype, possessed the IIB mitochondrial haplotype, and were mating type A1. Of the 16 isolates tested, all were determined to be metalaxyl resistant because isolates grown on 5 and 100 µg/ml metalaxyl exhibited growth greater than 40% of the unamended control. The 1995 outbreak was caused by the relatively rare US-7 genotype and started so late during the season that economic impact was minimal. Similarly, the 2006 outbreak was noted from only one commercial potato field at the time of harvest in September 2006. However, the genotype of the 2006 isolate has not been determined because the patch was destroyed before adequate samples could be collected. Because the disease occurs so sporadically in Alaska, fungicides are not routinely in use, but it is unlikely that the pathogen has persisted locally between outbreaks. The source of P. infestans is unknown for each of the occurrences in Alaska. However, possible routes include seed potatoes for home gardens or commercial farms, tomato transplants, and retail vegetables shipped to Alaska from out of state. While potato is Alaska's main vegetable crop, there are less than 405 ha (1,000 acres) of potatoes planted in the state, with the majority planted in the Matanuska Valley. To our knowledge, this is the first formal report of P. infestans on both tomato and commercial potato in Alaska. References: (1) E. K. Cash. Plant Dis. Rep. 20:121, 1936. (2) D. E. L. Cooke et al. Plant Pathol. 52:181, 2003. (3) S. B. Goodwin et al. Plant Dis. 79:1181, 1995. (4) G. W. Griffith and D. S. Shaw. Appl. Environ. Microbiol. 64:4007, 1998.

Dissipation of potassium and proton gradients inhibits mitochondrial hyperpolarization and cytochrome c release during neural apoptosis.

Exposure of rat hippocampal neurons or human D283 medulloblastoma cells to the apoptosis-inducing kinase inhibitor staurosporine induced rapid cytochrome c release from mitochondria and activation of the executioner caspase-3. Measurements of cellular tetramethylrhodamine ethyl ester fluorescence and subsequent simulation of fluorescence changes based on Nernst calculations of fluorescence in the extracellular, cytoplasmic, and mitochondrial compartments revealed that the release of cytochrome c was preceded by mitochondrial hyperpolarization. Overexpression of the anti-apoptotic protein Bcl-xL, but not pharmacological blockade of outward potassium currents, inhibited staurosporine-induced hyperpolarization and apoptosis. Dissipation of mitochondrial potassium and proton gradients by valinomycin or carbonyl cyanide p-trifluoromethoxy-phenylhydrazone also potently inhibited staurosporine-induced hyperpolarization, cytochrome c release, and caspase activation. This effect was not attributable to changes in cellular ATP levels. Prolonged exposure to valinomycin induced significant matrix swelling, and per se also caused release of cytochrome c from mitochondria. In contrast to staurosporine, however, valinomycin-induced cytochrome c release and cell death were not associated with caspase-3 activation and insensitive to Bcl-xL overexpression. Our data suggest two distinct mechanisms for mitochondrial cytochrome c release: (1) active cytochrome c release associated with early mitochondrial hyperpolarization, leading to neuronal apoptosis, and (2) passive cytochrome c release secondary to mitochondrial depolarization and matrix swelling.

Up-regulation of Bcl-xL in response to subtoxic beta-amyloid: role in neuronal resistance against apoptotic and oxidative injury.

Neuron death in Alzheimer's disease is believed to be triggered by an increased production of amyloidogenic beta-amyloid peptides, involving both increased oxidative stress and activation of a conserved death program. Bcl-xL, an anti-apoptotic protein of the Bcl-2 family, is expressed at high levels in the adult nervous system. Exposure of neuronal cultures to subtoxic concentrations of beta-amyloid peptide 1-40 (1-10microM) or the fragment 25-35 (1-10microM) up-regulated both bcl-xL mRNA and Bcl-xL protein levels, determined by reverse transcriptase-polymerase chain reaction and western blot analysis. Bcl-xL protein was also up-regulated during oxidative stress induced by exposure to hydrogen peroxide (3-100microM) or ferric ions (1-10microM). In contrast, apoptotic stimuli (exposure to staurosporine or serum withdrawal) actually decreased neuronal Bcl-xL expression. To investigate the role of Bcl-xL in cell death relevant to Alzheimer's disease, we stably overexpressed Bcl-xL in human SH-SY5Y neuroblastoma cells. Cells overexpressing Bcl-xL were significantly protected from beta-amyloid neurotoxicity and staurosporine-induced apoptosis compared to vector-transfected controls. In contrast, Bcl-xL overexpression only conferred a mild protection against oxidative injury induced by hydrogen peroxide. We conclude that up-regulation of Bcl-xL expression in response to subtoxic concentrations of beta-amyloid is a stress response that increases the resistance of neurons to beta-amyloid neurotoxicity primarily by inhibiting apoptotic processes.

Radiographic findings in osteoarthritis of the knee joint are not correlated with cartilage histomorphology or immunohistochemistry.

The purpose of this study was to investigate whether radiographic joint space narrowing (JSN) of the lateral knee compartment predicts the histomorphological or immunhistochemical grading in cases of osteoarthritis of the knee joint. The lateral joint space was measured on weight-bearing radiographs. Femoral osteochondral plugs of 29 patients undergoing total knee replacement were obtained from lateral condyles. All these patients had severe osteoarthritis of the medial compartment, with the lateral compartment showing different stages of osteoarthritis. The specimens were histomorphologically evaluated with the Mankin score, and the expression of the cartilage-degrading enzymes MMP1 and MMP3 was measured. There was no correlation between the joint space and histomorphological or immunohistochemical data, whereas the enzyme expression was correlated with histomorphological grading. We conclude that radiographic assessment alone is not sufficient to evaluate the cartilage damage of the lateral condyle.

Delayed mitochondrial dysfunction in excitotoxic neuron death: cytochrome c release and a secondary increase in superoxide production.

An increased production of superoxide has been shown to mediate glutamate-induced neuron death. We monitored intracellular superoxide production of hippocampal neurons during and after exposure to the glutamate receptor agonist NMDA (300 microm). During a 30 min NMDA exposure, intracellular superoxide production increased significantly and remained elevated for several hours after wash-out of NMDA. After a 5 min exposure, superoxide production remained elevated for 10 min, but then rapidly returned to baseline. Mitochondrial membrane potential also recovered after wash-out of NMDA. However, recovery of mitochondria was transient and followed by delayed mitochondrial depolarization, loss of cytochrome c, and a secondary rise in superoxide production 4-8 hr after NMDA exposure. Treatment with a superoxide dismutase mimetic before the secondary rise conferred the same protection against cell death as a treatment before the first. The secondary rise could be inhibited by the complex I inhibitor rotenone (in combination with oligomycin) and mimicked by the complex III inhibitor antimycin A. To investigate the relationship between cytochrome c release and superoxide production, human D283 medulloblastoma cells deficient in mitochondrial respiration (rho(-) cells) were exposed to the apoptosis-inducing agent staurosporine. Treatment with staurosporine induced mitochondrial release of cytochrome c, caspase activation, and cell death in control and rho(-) cells. However, a delayed increase in superoxide production was only observed in control cells. Our data suggest that the delayed superoxide production in excitotoxicity and apoptosis occurs secondary to a defect in mitochondrial electron transport and that mitochondrial cytochrome c release occurs upstream of this defect.

Activation of calpain I converts excitotoxic neuron death into a caspase-independent cell death.

Glutamate receptor overactivation contributes to neuron death after stroke, trauma, and epileptic seizures. Exposure of cultured rat hippocampal neurons to the selective glutamate receptor agonist N-methyl-d-aspartate (300 microm, 5 min) or to the apoptosis-inducing protein kinase inhibitor staurosporine (300 nm) induced a delayed neuron death. In both cases, neuron death was preceded by the mitochondrial release of the pro-apoptotic factor cytochrome c. Unlike staurosporine, the N-methyl-d-aspartate-induced release of cytochrome c did not lead to significant activation of caspase-3, the main caspase involved in the execution of neuronal apoptosis. In contrast, activation of the Ca(2+)-activated neutral protease calpain I was readily detectable after the exposure to N-methyl-d-aspartate. In a neuronal cell-free apoptosis system, calpain I prevented the ability of cytochrome c to activate the caspase cascade by inhibiting the processing of procaspase-3 and -9 into their active subunits. In the hippocampal neuron cultures, the inhibition of calpain activity restored caspase-3-like protease activity after an exposure to N-methyl-d-aspartate. Our data demonstrate the existence of signal transduction pathways that prevent the entry of cells into a caspase-dependent cell death program after the mitochondrial release of cytochrome c.

Mitochondrial depolarization is not required for neuronal apoptosis.

Mitochondria are sites of cellular energy production but may also influence life and death decisions by initiating or inhibiting cell death. Mitochondrial depolarization and the subsequent release of pro-apoptotic factors have been suggested to be required for the activation of a cell death program in some forms of neuronal apoptosis. We induced apoptosis in cultured rat hippocampal neurons by exposure to the protein kinase inhibitor staurosporine (STS) (300 nM). The time course of mitochondrial membrane potential (DeltaPsi(m)) during apoptosis was examined using the probe tetramethylrhodamine ethyl ester (TMRE). Cells exhibited no decrease in TMRE fluorescence, indicative of mitochondrial depolarization, up to 8 hr after STS exposure. Rather, baseline TMRE fluorescence remained unchanged up to 2 hr and thereafter actually increased significantly. Throughout this time period, the mitochondria could also be depolarized with the protonophore carbonyl cyanide p-trifluoromethoxy-phenylhydrazone (FCCP, 0.1 microM), exhibiting the same relative magnitude of fluorescence release (unquenching) as controls. Even after 16 hr of staurosporine treatment, neurons that showed signs of nuclear apoptosis maintained DeltaPsi(m) and could be depolarized with FCCP. In contrast, caspase-3-like activity had increased roughly sevenfold by 2 hr and >20-fold by 8 hr. Double-labeling of hippocampal neurons with the potential-sensitive probe Mitotracker Red Chloromethyl X-Rosamine and an antibody to cytochrome c demonstrated at the subcellular level that mitochondrial cytochrome c release also occurred in the absence of mitochondrial depolarization. These data suggest that mitochondrial depolarization is not a decisive event in neuronal apoptosis.

Staurosporine-induced apoptosis of cultured rat hippocampal neurons involves caspase-1-like proteases as upstream initiators and increased production of superoxide as a main downstream effector.

We induced apoptosis in cultured rat hippocampal neurons by exposure to the protein kinase inhibitor staurosporine (30 nM, 24 hr). Treatment with the antioxidant (+/-)-alpha-tocopherol (100 microM) or the superoxide dismutase-mimetic manganese tetrakis (4-benzoyl acid) porphyrin (1 microM) significantly reduced staurosporine-induced cell death. Using hydroethidine-based digital videomicroscopy, we observed a significant increase in intracellular superoxide production that peaked 6-8 hr into the staurosporine exposure. This increase occurred in the absence of gross mitochondrial depolarization monitored with the voltage-sensitive probe tetramethylrhodamine ethyl ester. We then prepared extracts from staurosporine-treated hippocampal neurons and monitored cleavage of acetyl-Tyr-Val-Ala-Asp-aminomethyl-coumarin and acetyl-Asp-Glu-Val-Asp-AMC, fluorogenic substrates for caspase-1-like and caspase-3-like proteases, respectively. Staurosporine caused a significant increase in caspase-1-like activity that preceded intracellular superoxide production and reached a maximum after 30 min. Caspase-3-like activity paralleled intracellular superoxide production, with peak activity seen after 8 hr. Treatment with the corresponding caspase-3-like protease inhibitor acetyl-Asp-Glu-Val-Asp-aldehyde (10 microM) prevented the increase in caspase-3-like activity and staurosporine-induced nuclear fragmentation, but failed to prevent the rise in superoxide production and subsequent cell death. In contrast, treatment with caspase-1-like protease inhibitors reduced both superoxide production and cell death. Of note, antioxidants prevented superoxide production, caspase-3-like protease activity, and cell death even when added 4 hr after the onset of the staurosporine exposure. These results suggest a scenario of an early, caspase-1-like activity followed by a delayed intracellular superoxide production that mediates staurosporine-induced cell death of cultured rat hippocampal neurons.

Characterization of human immunodeficiency virus type 1 mutants with decreased sensitivity to proteinase inhibitor Ro 31-8959.

A human immunodeficiency virus type 1 (HIV-1) variant with highly reduced susceptibility to Ro 31-8959, an inhibitor of the viral proteinase, has been selected by repeated passage of wild-type virus in CEM cells in the presence of increasing concentrations of the inhibitor. Peptide sequences of the proteinase of selected virus were obtained from proviral DNA. Sequence comparison to wild-type (wt) proteinase demonstrated two amino acid substitutions in the resistant virus, a Gly to Val exchange at position 48 and a Leu to Met exchange at position 90. Furthermore, sequences of intermediate passage virus suggest contributions from positions 12, 36, 57, and 63 in early steps of resistance development. The selected virus showed a ca. 40-fold increase in 50% inhibitory concentration of Ro 31-8959. Growth kinetics of resistant virus were comparable to wild-type virus and the resistant genotype proved to be stable in the absence of inhibitor. Directed mutagenesis of the HIV-1 HXB2 proteinase at positions 48 and 90 suggested that each mutation alone led to a moderate decrease in sensitivity of the recombinant virus to proteinase inhibitor. However, a recombinant virus carrying both mutations in the proteinase gene showed a significant reduction in its sensitivity to Ro 31-8959 thus proving the importance of these exchanges for the resistance phenotype.

[Studies on the mobility of soft tissue marks of the lower face]. / Untersuchugen zur Mobilität von markierten Weichteilpunkten im Untergesicht.

The mobility of soft tissue points used for measuring the rest vertical dimension was assessed in 29 probands with full dentitions and 24 edentulous subjects. A video-aided procedure was used for recording the measuring pictures. Similar results were obtained with fully dentulous and edentulous subjects. For the upper jaw the point of the nose was the mark undergoing the least fluctuations, due to the action of the mimic muscles. For the lower jaw the least mobile skin area was between the labiomental sulcus and the gnathion.

Rational design of peptide-based HIV proteinase inhibitors.

A series of peptide derivatives based on the transition-state mimetic concept has been designed that inhibit the proteinase from the human immunodeficiency virus (HIV). The more active compounds inhibit both HIV-1 and HIV-2 proteinases in the nanomolar range with little effect at 10 micromolar against the structurally related human aspartic proteinases. Proteolytic cleavage of the HIV-1 gag polyprotein (p55) to the viral structural protein p24 was inhibited in chronically infected CEM cells. Antiviral activity was observed in the nanomolar range (with one compound active below 10 nanomolar) in three different cell systems, as assessed by p24 antigen and syncytium formation. Cytotoxicity was not detected at 10 and 5 micromolar in C8166 and JM cells, respectively, indicating a high therapeutic index for this new class of HIV proteinase inhibitors.

Microchemical synthesis of the serotonin receptor ligand, 125I-LSD.

The synthesis and properties of 2-[125I]-lysergic acid diethylamide, the first 125I-labeled serotonin receptor ligand, are described. A novel microsynthesis apparatus was developed for this synthesis. The apparatus employs a micromanipulator and glass micro tools to handle microliter to nanoliter volumes on a microscope stage. This apparatus should be generally useful for the synthesis of radioligands and other compounds when limited amounts of material must be handled in small volumes.

[(125)I]LSD binding to serotonin and dopamine receptors in bovine caudate membranes.

[(125)I]LSD (labeled at the 2 position) has been introduced as the first (125)I-labeled ligand for serotonin 5-HT(2) (S2) receptors. In the present study we examined the binding of [(125)I]LSD and its non-radioactive homologue, 2I-LSD, to bovine caudate homogenates. The binding of [(125)I]LSD is saturable, reversible, stereospecific and is destroyed by boiling the membranes. The specific to total binding ratio in this tissue is 75-80% and Scatchard plots of the binding data reveal K(d) = 1.1 nM, B(max) = 9.6 fmol/mg wet weight tissue. The association and dissociation rate constants are highly temperature dependent. At 0 degrees C the net dissociation is less than 5% after 1 h and the association rate is proportionately slow. IC(50) values for a variety of compounds show a clear 5-HT(2) (S2) serotonergic pattern at this [(125)I]LSD site. Blockage of this primary 5-HT(2) (S2) caudate binding site by 0.3 ?M mianserin reveals the presence of a weaker [(125)I]LSD binding site with a K(d) = 9.1 nM, B(max) = 7.6 fmol/mg tissue. This secondary site is a D3 dopaminergic receptor site, as shown by the relative abilities of various displacers to inhibit this binding. Binding studies with nonradioactive 2I-LSD reveal a clear preference for D2 over D3 dopamine receptor sites. [(125)I]LSD is a sensitive and selective label for 5-HT(2) (S2) serotonin receptor sites in both rat frontal cortex and bovine caudate membranes. Blockage of the primary bovine caudate [(125)I]LSD binding site with mianserin allows the high sensitivity of [(125)I]LSD to be applied to D2 dopamine receptor studies as well.

Characterization of 125I-lysergic acid diethylamide binding to serotonin receptors in rat frontal cortex.

125I-Lysergic acid diethylamide (125I-LSD) is the first 125I-labeled ligand for serotonin receptor studies. Its binding to rat frontal cortex membranes is saturable, reversible, and stereospecific. Specific binding is linearly dependent on tissue concentration and represents 70-80% of the total binding. Scatchard plots of the binding data are linear with a KD of 1.5 nM, a Bmax of 12.4 fmol/mg wet weight tissue, and a Hill slope of 1.02. The binding kinetics are highly temperature-dependent. At 37 degrees C the bimolecular association rate constant is 1.28 X 10(8) min-1 M-1 and the dissociation rate constant is 0.087 min-1 (t 1/2 = 8.0 min). At 0 degrees C less than 4% dissociation occurs over 40 min and the association rate is similarly depressed. Inhibition of 125I-LSD binding by a variety of serotonergic, dopaminergic, and adrenergic ligands reveals a 5-hydroxytryptamine2 (5-HT2) serotonergic profile for this binding site. Regional distribution studies of 125I-LSD binding in rat brain show that areas with the highest levels of binding include the cortex and striatum. Iodinated radioligands can be synthesized with specific activities exceeding 2,000 Ci/mmol, which makes them approximately 75-fold more sensitive than tritiated radioligands. This high specific activity, coupled with the selectivity of 125I-LSD for 5-HT2 sites, makes this ligand a sensitive new probe for 5-HT2 serotonin receptors.

New potent inhibitors of angiotensin converting enzyme.

Using an earlier model of the favoured orientation of binding functions of angiotensin converting enzyme (ACE) inhibitors, it has been possible to postulate a new, 7,6-bicyclic system, based on hexahydropyridazine, which might be expected to have high potency. Some members of this system which have been synthesised have been shown to be very active ACE inhibitors, in vitro and in vivo.