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.

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.

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.

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.

The design of a new group of angiotensin-converting enzyme inhibitors.

Using X-ray and NMR data relating to the conformation of the antihypertensive, angiotensin-converting enzyme inhibitor, captopril, and structure--activity relationships of analogues, it has been possible to postulate with the aid of computer graphics, the orientation of the three functions, the thiol, the terminal carboxyl and the carbonyl group which are involved in binding to the enzyme. Bicyclic mimetics of captopril, with related arrays of these functions, have been designed and synthesized. Compounds with the closest approximation to the array in captopril are the most active inhibitors of angiotensin converting enzyme, in vitro.

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.

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.

[(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.

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.

The nature of the Oedipus complex in the Dora case.

In this paper we sought to study, through a detailed review of Dora's analytic material, the specific nature of her oedipal conflict and its expression in her treatment with Freud as manifested in her dreams, associations, and transference. We found that while Freud was surely correct in considering Dora's positive oedipal conflict as an important source of her psychopathology, the implication of her early regression to a phallic-oedipal position was not fully appreciated or interpreted, leading to her eventual premature termination. While Freud analyzed, largely following the end of the treatment, Dora's erotic love for Frau K., he did not see this love as deriving from an integrated phallic-oedipal complex which included competitive and castrative strivings toward men as well as wishes to love a woman from a phallic position. Thus we sought to extend Freud's analysis of the case by delineating the specific form of Dora's homosexual love, the genetic sources for it, and the aggressive wish toward men as they derive from a particular substage of the oedipal phase for females, the phallic-oedipal phase. We conclude that Dora's hostility toward Freud, her father and Herr. K. resulted not only from injured pride but, more basically, from her jealousy and rivalry with father for mother's love. In her treatment Dora's phallic-oedipal wishes became increasingly dominant. They were manifested in the transference with her first acting to engage Freud's interest and then thwarting his interpretive efforts, ultimately by leaving the treatment altogether. The case well illustrates the importance of understanding the role of the phallic-oedipal phase in female hysterical patients.

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.

Psychological effects of symptoms associated with labyrinthitis. a case study.

The psychological effects of postviral labyrinthitis in a patient who had been undergoing intensive psychotherapy are reported. The mildness of the physiological symptome was in contrast to the magnitude of the psychological consequences. The general psychological effects of illness on the patient are differentiated from the specific psychological correlates of vertiginous symptoms. The factors that contributed to the psychological effects and the psychological impact of these symptoms are discussed. There is a special potency to the cited effects in patients with specific personality characteristics.

Some clinical manifestations of structural defects in a borderline personality.

A case is presented that illustrates specific clinical manifestations of structural deficits and psychopathological complexes characteristic of borderline patients. Following an overview of the patient's presenting problems and history, the patient's personality organization is conceptualized metapsychologically. Then the clinical manifestations of three core problem areas in the patient are explored: (1) deficits in narcissistic cathexis leading to clinically apparent, primitive, self-confirming character traits, (2) fear of helplessness and pathological attempts to establish a sense of control, and (3) arrest of drive development coupled with limited ego capacity for sublimation.