Preventing nanoscale wear of atomic force microscopy tips through the use of monolithic ultrananocrystalline diamond probes.

Nanoscale wear is a key limitation of conventional atomic force microscopy (AFM) probes that results in decreased resolution, accuracy, and reproducibility in probe-based imaging, writing, measurement, and nanomanufacturing applications. Diamond is potentially an ideal probe material due to its unrivaled hardness and stiffness, its low friction and wear, and its chemical inertness. However, the manufacture of monolithic diamond probes with consistently shaped small-radius tips has not been previously achieved. The first wafer-level fabrication of monolithic ultrananocrystalline diamond (UNCD) probes with <5-nm grain sizes and smooth tips with radii of 30-40 nm is reported, which are obtained through a combination of microfabrication and hot-filament chemical vapor deposition. Their nanoscale wear resistance under contact-mode scanning conditions is compared with that of conventional silicon nitride (SiN(x)) probes of similar geometry at two different relative humidity levels (approximately 15 and approximately 70%). While SiN(x) probes exhibit significant wear that further increases with humidity, UNCD probes show little measurable wear. The only significant degradation of the UNCD probes observed in one case is associated with removal of the initial seed layer of the UNCD film. The results show the potential of a new material for AFM probes and demonstrate a systematic approach to studying wear at the nanoscale.

Antidiarrheal activity of aqueous extract of Hermannia incana Cav. leaves in Wistar rats.

Hermannia incana Cav. is a prostrate herb used to treat diarrhea, stomach ache, nausea and vomiting, by the people of Eastern Cape Province, South Africa. The phytochemical screening as well as the antidiarrheal activity of H. incana leaf extract at 200, 400 and 600 mg/kg body weight was evaluated in rats. Phytochemical screening revealed the presence of bioactive agents such as alkaloids, tannins, saponins, phenolics, triterpenes, cardiac glycosides, flavonoids, cardenolides and dienolides. The extract significantly prolonged the time of induction of diarrhea, reduced the frequency of diarrheal episodes and water content of the feces, and inhibited castor oil-induced enteropooling. The extract also suppressed intestinal propulsive movement of a charcoal meal through the gastrointestinal tract. These results demonstrate the antidiarrheal properties of the extract, thereby supporting the folkloric use of the plant as an antidiarrheal agent in the Eastern Cape of South Africa.

Molecular genetics of tomato fruit ripening.

Tomato ripening is an excellent system for studying control of gene expression in plants. A multiplicity of well-defined biochemical and genetic changes occur in a precise sequence, regulated by a gaseous hormone. The generation of targeted mutations using sense and antisense genes provides a means of manipulating endogenous gene expression, both for answering fundamental questions and for crop improvement.

Effects of plasmin on von Willebrand factor multimers. Degradation in vitro and stimulation of release in vivo.

von Willebrand factor (vWF), a multimeric protein that mediates platelet adhesion, circulates in association with the procoagulant Factor VIII (FVIII). In previous reports, plasmin was shown in vitro to inactivate FVIII and cleave the vWF subunit extensively, but to cause only a modest decrease in vWF platelet-agglutinating activity. In the present study, the digestion of vWF multimers by plasmin was analyzed by sodium dodecyl sulfate-agarose gel electrophoresis and radioimmunoblotting. In vitro, plasmin degraded the large vWF multimers to smaller forms that could be distinguished from the small multimers present before digestion only by a slightly increased electrophoretic mobility. These plasmin-cleaved "multimers" were composed of disulfide-linked fragments with no intact vWF subunits. Thus, many plasmin cleavages occur within disulfide loops. The slight increase in mobility of plasmin-digested vWF is in part explained by the early cleavage from the multimers of a 34,000-mol wt peptide, which was purified and partially sequenced. The amino-terminal sequence (33 residues) agrees with the previously reported sequence (15 residues) for the amino terminus of the intact vWF subunit. Analysis of plasmin-digested vWF allowed deduction of a model for the native vWF structure, including the approximate location of the interprotomer disulfide bond(s). To determine whether plasmin would digest vWF in vivo, plasmas from 12 patients and 2 normal volunteers who received intravenous streptokinase (SK) were analyzed. Rather than vWF digestion, a two- to threefold rise in vWF antigen and platelet-agglutinating activity occurred within 2 h after a single SK dose, and the increase was greatest among the largest multimers. In contrast, FVIII clotting activity dropped to 10-20% of pre-SK levels. Thus, although plasmin destroys FVIII, a pharmacologically induced fibrinolytic state is associated with significant release of vWF from endothelial cells, platelets, or some other storage pool.

Functional improvement of jeopardized myocardium following intracoronary streptokinase infusion in acute myocardial infarction.

The effect of reperfusion on regional left ventricular performance following acute myocardial infarction in man was determined. Intracoronary streptokinase was administered in 24 patients within 6 h of the onset of symptoms. 15 patients (62%) were successfully recanalized during the initial study. Mean percent radial shortening (%RS) in both the jeopardized and compensatory regions were determined using 23 radii from the centroid of diastolic and systolic angiographic silhouettes. Sequential measurements were obtained during repeat cardiac catheterization studies at 24 h in 19 patients and before discharge from the hospital (16 +/- 11 d) in 15 patients. At the time of the predischarge study, each acutely reperfused patient showed improvement in %RS in the jeopardized region (P = 0.01) with 56% returning to the normal range. Despite the uniform improvement in the contractile function of the jeopardized region in each reperfused patient, the global ejection fraction showed no improvement or a decrease at the time of the chronic study in 44%. This was due to a decrease in the compensatory wall motion in the uninvolved segments between the acute and chronic study in each case. Neither the %RS nor the ejection fraction changed significantly at the time of the chronic study in the patients who could not be acutely recanalized. These data indicate (a) significant salvage of jeopardized myocardium associated with recovery of contractile function in patients reperfused during the first 6 h of chest pain following acute myocardial infarction; (b) no improvement in regional or global left ventricular performance in patients who could not be reperfused acutely; and (c) the ejection fraction is strongly influenced by changes in the compensatory wall motion of the uninvolved segments and does not accurately reflect changes in the contractile function of the jeopardized myocardium.

In vitro phototoxicity of glycoconjugated porphyrins and chlorins in colorectal adenocarcinoma (HT29) and retinoblastoma (Y79) cell lines.

BACKGROUND: Retinoblastoma is the most common malignant intraocular tumor in children. The current treatment gives a good vital prognostic but there are several drawbacks to the arsenal of "classical antitumoral" therapies. Photodynamic therapy (PDT) could be an exciting non-toxic and non-mutagenic alternative protocol. METHOD: In this paper, we report about the screening of the in vitro photocytotoxicity of hydrophenylporphyrins and chlorins and their glycoconjugated derivatives in a human retinoblastoma cell line (Y79) and for comparison in a colorectal adenocarcinoma cell line (HT29). RESULTS: Despite lower photodynamic activity than that observed for hydroxylated photosensitizers, in particular Foscan(®) glycoconjugated derivatives display phototoxicity (IC50 2.4-0.05µM ±10%) against Y79 cells with examples of significant intrinsic cytotoxicity. Amongst them the triglucosyl porphyrin 10 is highly photocytotoxic (IC50 0.9µM ±10%) but is fully devoid of cytotoxicity (IC50>15µM). The photoactivity is highly modulated by the presence of a diethyleneglycol spacer between the chromophore and the glycoside (compounds 14-17, IC50 0.5, 0.6, 0.05 and 0.35µM ±10%) and by the anomeric configuration of the sugar (compound 15 and 17, IC50 0.6 and 0.05µM ±10% respectively). One of the main problems for the use of Foscan(®) is its poor solubility which might be improved by glycoconjugation. Moreover Foscan has been shown to induce necrosis after PDT leading to a possible ulceration of surrounding tissues unsuitable for a conservative treatment. A preferential mitochondrial subcellular localization which has been previously reported for some glycoconjugated photosensitizers could enhance the contribution of apoptosis process. CONCLUSION: Tri-α-O-galactosyl porphyrin 16 is a better candidate than Foscan(®) for a clinical application of PDT for a conservative therapy of retinoblastoma.

Plants genetically modified to produce N-acylhomoserine lactones communicate with bacteria.

N-acylhomoserine lactones (AHLs) play a critical role in plant/microbe interactions. The AHL, N-(3-oxohexanoyl)-L-homoserine lactone (OHHL), induces exoenzymes that degrade the plant cell wall by the pathogenic bacterium Erwinia carotovora. Conversely, the antifungal activity of the biocontrol bacterium Pseudomonas aureofaciens 30-84 is due (at least in part) to phenazine antibiotics whose synthesis is regulated by N-hexanoylhomoserine lactone (HHL). Targeting the product of an AHL synthase gene (yenI) from Yersinia enterocolitica to the chloroplasts of transgenic tobacco plants caused the synthesis in plants of the cognate AHL signaling molecules (OHHL and HHL). The AHLs produced by the transgenic plants were sufficient to induce target gene expression in several recombinant bacterial AHL biosensors and to restore biocontrol activity to an HHL-deficient P. aureofaciens strain. In addition, pathogenicity was restored to an E. carotovora strain rendered avirulent as a consequence of a mutation in the OHHL synthase gene, carI. The ability to generate bacterial quorum-sensing signaling molecules in the plant offers novel opportunities for disease control and for manipulating plant/microbe interactions.

Volatile components from the roots of Solanum pseudocapsicum.

The volatile components obtained by hydrodistillation of Solanum pseudocapsicum roots were analyzed by gas chromatography-mass spectrometry. A total of 41 compounds, representing 50% of the oil, were identified. The oil was found to contain fatty acids (26.8%), terpenoids (7.6%), and aldehydes (5.3%) as the major components. The dominant compounds were hexadecanoic acid (24.1%), 2-methoxy-3-isopropylpyrazine (2.8%), and 15-methylhexadecanoic acid (2.1%). Other notable components include beta-elemene and delta-elemene. The high proportion of fatty acids in this plant could contribute to its medicinal properties.

Bioactive sesquiterpene lactones from the leaves of Vernonia amygdalina.

Phytochemical analysis of the leaves of Vernonia amygdalina yielded two known sesquiterpene lactones: vernolide and vernodalol. The two compounds were tested by agar dilution method against 10 bacteria strains and 5 fungi species. Both compounds exhibited a significant bactericidal activity against five Gram positive bacteria while lacking efficacy against the Gram negative strains. In the antifungal test, while vernolides exhibited high activity with LC(50) values of 0.2, 0.3 and 0.4 mg/ml against Penicillium notatum, Aspergillus flavus, Aspergillus niger and Mucor hiemalis, respectively, vernodalol showed moderate inhibitions against Aspergillus flavus, Penicillium notatum and Aspergillus niger with LC(50) values of 0.3, 0.4 and 0.5 mg/ml, respectively. Both compounds were ineffective against Fusarium oxysporum, a microbe known to be highly resistant to chemical agents. However, the antimicrobial results of this study correspond positively with the claimed ethnomedical uses of the leaves of Vernonia amygdalina in the treatment of various infectious diseases.

Ribonucleic acid from the higher plant Matthiola incana. Molecular weight measurements and DNA-RNA hybridisation studies.

The percentage of DNA from the crucifer Matthiola incana coding for different types of RNA was measured by filter saturation hybridisation experiments using RNA labelled in vivo. In addition, the melting curves of the various DNA - RNA hybrids formed and the buoyant densities of the DNA sequences complementary to different types of RNA were measured. 1. The RNA preparations used were 25, 18, and 5 S rRNA and 4 S RNA, purified by gel electrophoresis, and poly(A)-containing RNA purified by oligo-(dT)-cellulose chromatography. The molecular weights of the 25 S and 18 S rRNAs, calculated from the mobility in formamide-acrylamide gels relative to Escherichia coli RNA, are 1.25 - 10(6) and 0.64 - 10(6). The rRNA precursor has a molecular weight of approx. 2.1 - 10(6) and the average molecular weight of the poly(A)-containing RNA from both cotyledons and roots is 4 - 10(5). 2. The percentage of the genome, calculated on the basis of double-stranded DNA, coding for these RNAs and the estimated number of genes per haploid DNA amount are approximately 0.46% and 1100 for 25 S plus 18 S rRNA, 0.032% and 3600 for 5 S rRNA and 0.072% and 13 000 for 4 S RNA. In filter hybridisation experiments very little hybridisation of poly(A)-containing RNA was found. A rapidly-hybridising component is attributed to small amounts of contaminating rRNA. 3. M. incana DNA has a main band at 1.697 g - ml-1 in CsCl and a satellite constituting approximately 3% of the DNA, at 1.708 g - ml-1 - 25 and 18 S rRNA hybridise to DNA with a buoyant density of 1.701--2 g - ml-1. The buoyant density of 5 S DNA is slightly less at 1.700--1 g - ml-1. 4. S RNA hybridises to at least two separate regions, one within the main-band DNA and a second lighter component. None of the RNAs tested hybridised to the satellite DNA. The Tm of the DNA - RNA hybrids in 1 X SSC is 89 degrees C for 25 S rRNA, 85 degrees C for 5 S rRNA and 82 degrees C for 4 S RNA. 4. 5 and 4 S RNA preparations contain fragments which hybridise to sequences complementary to high-molecular-weight rRNA. This spurious hybridisation can be eliminated by competition with unlabelled high-molecular-weight RNA.

Isolation and characterisation of 14-S RNA from spinach chloroplasts.

14-S RNA was purified from spinach chloroplasts. It has a molecular weight of 0.43 . 10(6) and the following nucleotide composition: 20% CMP, 23.9% AMP, 24.2% GMP and 31.9% UMP. The accumulation of 14-S RNA in chloroplasts of cotyledons of dark-grown plants is stimulated by light. Conditions are described for the isolation of 14-S RNA in the absence of appreciable fragmentation of chloroplast 23-S rRNA and the evidence that it represents a distinct type of chloroplast RNA is discussed. Translation of 14-S RNA in a protein synthesising system from Escherichia coli gives rise to two polypeptides with molecular weights of 13 200 and 12 600 and the possible role of 14-S RNA as a chloroplast messenger is discussed.

Preformed poly(A)-containing RNA in zoospores of Blastocladiella emersonii.

Discharged zoospores of Blastocladiella emersonii contain poly(A)-containing RNA, which is performed during the initial stages of zoospore differentiation in the sporangium and is stable in vivo for at least 4 h. The average molecular weight of this RNA fraction is estimation to be 5.5-10(5) or 1600 nucleotides. The average length of the poly(A) sequence is approx. 100 nucleotides Poly(A)-containing RNA does not appear to accumulate in a gamma-particle fraction previously suggested to be involved in the storage of mRNA.

Molecular and genetic characterization of a novel pleiotropic tomato-ripening mutant

In this paper we describe a novel, dominant pleiotropic tomato (Lycopersicon esculentum)-ripening mutation, Cnr (colorless nonripening). This mutant occurred spontaneously in a commercial population. Cnr has a phenotype that is quite distinct from that of the other pleiotropic tomato-ripening mutants and is characterized by fruit that show greatly reduced ethylene production, an inhibition of softening, a yellow skin, and a nonpigmented pericarp. The ripening-related biosynthesis of carotenoid pigments was abolished in the pericarp tissue. The pericarp also showed a significant reduction in cell-to-cell adhesion, with cell separation occurring when blocks of tissue were incubated in water alone. The mutant phenotype was not reversed by exposure to exogenous ethylene. Crosses with other mutant lines and the use of a restriction fragment length polymorphism marker demonstrated that Cnr was not allelic with the pleiotropic ripening mutants nor, alc, rin, Nr, Gr, and Nr-2. The gene has been mapped to the top of chromosome 2, also indicating that it is distinct from the other pleiotropic ripening mutants. We undertook the molecular characterization of Cnr by examining the expression of a panel of ripening-related genes in the presence and absence of exogenous ethylene. The pattern of gene expression in Cnr was related to, but differed from, that of several of the other well-characterized mutants. We discuss here the possible relationships among nor, Cnr, and rin in a putative ripening signal cascade.

Pharmacokinetics of streptokinase in patients based on amidolytic activator complex activity.

The purpose of this study was to determine the pharmacokinetics of intravenous streptokinase in humans. Five patients with myocardial infarction, six patients with venous thromboembolism, and two normal volunteers were studied. The patients with myocardial infarction received 500,000 U over 30 minutes, the patients with venous thromboembolism received 250,000 U over 30 minutes followed by 100,000 U/hr over 16 to 78 hours, and the normal volunteers received 100,000 U over 15 minutes. Plasma streptokinase levels were measured based on the amidolytic activity of the streptokinase-plasminogen complex on the chromogenic substrate S-2251. Pharmacokinetic parameters were: biologic half-life 82 +/- 25 minutes, total clearance 10.8 +/- 8.8 ml/min, and apparent volume of distribution 1.10 +/- 0.71 l. Streptokinase levels declined progressively during the continuous, prolonged infusion in the patients with venous thromboembolism over 60 hours of treatment. We conclude that there are distinct time-dependent changes in the pharmacokinetics of streptokinase during continuous intravenous infusion and that this phenomenon is likely to be associated with progressively decreasing thrombolytic efficacy.

3D-QSAR CoMFA on cyclin-dependent kinase inhibitors.

Several series of cyclin-dependent kinase inhibitors previously prepared in our laboratory were compared using 3D-QSAR (CDK1) and docking (CDK2) techniques. Evaluation of our own library of 93 purine derivatives served to establish the model which was validated by evaluation of an external library of 71 compounds. The best predictions were obtained with the CoMFA standard model (q(2) = 0.68, r(2) = 0.90) and with the CoMSIA combined steric, electrostatic, and lipophilic fields (q(2) = 0.74, r(2) = 0.90). The CDK1 3D-QSAR model was then superimposed to the ATP/CDK2 binding site, giving direct contour maps of the different fields. Although too few compounds were evaluated on CDK5 to derive a 3D-QSAR model, some interesting SARs have been deduced. Comparison of the results obtained from both methods helped with understanding the specific activity of some compounds and designing new specific CDK inhibitors.

A 3D QSAR study of a series of HEPT analogues: the influence of conformational mobility on HIV-1 reverse transcriptase inhibition.

Quantitative structure-activity relationships (QSAR) have been established for 87 analogues of 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT), a potent inhibitor of the HIV-1 reverse transcriptase (RT). Of these 87 nonnucleoside RT inhibitors, 9 novel HEPT analogues were used in the study and the others were taken from the literature. The predictive ability of these relationships has been evaluated using a large set of 54 compounds which were not used to derive the activity model. Descriptors related to the conformational changes were found to be an important factor which underlies RT inhibitory activity in the HEPT series. Indeed, the QSAR model provides evidence concerning the conformational transformations the molecules may undergo during the inhibition process. The established relationships are supplementary to the experimental study on the binding of HEPT type inhibitors to RT by Hopkins et al. (J. Med. Chem. 1996, 39, 1589-1600). The present study suggests a quantitative interpretation of the structure-activity relationships which otherwise cannot be explained within the framework of the crystal inhibitor-protein model. This information is pertinent to the further design of new HEPT type RT inhibitors.

Synthesis and anti-HIV activity of novel N-1 side chain-modified analogs of 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT).

A series of 33 N-1 side chain-modified analogs of 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine (1, HEPT) were synthesized and evaluated for their anti-HIV-1 activity. In particular, the influence of substitution of the terminal hydroxy group of the acyclic structure of HEPT and the structural rigidity of this side chain were investigated. Halo (7, 8), azido (9), and amino (10-15) derivatives were synthesized from HEPT via the p-tosylate derivative 6. Acylation of the primary amine 15 afforded the amido analogs 16-20. The diaryl derivatives 26-29 were prepared by reaction of HEPT, or of the 6-(2-pyridylthio) analog 23, with diaryl disulfides in the presence of tri-n-butylphosphine. Compounds 39-41, in which the N-1 side chain is rigidified by incorporation of an E-configured double bond, were obtained by palladium(0)-catalyzed coupling of several different 6-(arylthio)uracil derivatives (37, 38) with allyl acetates 33. Compounds 13, 40a,c,d,f, and 41, incorporating an aromatic ring at the end of the acyclic side chain, were found to be more potent than the known diphenyl-substituted HEPT analog BPT (2), two of them, 40c,d, being 10-fold more active.

Preparation and anti-HIV activity of N-3-substituted thymidine nucleoside analogs.

A series of 22 derivatives of AZT substituted at the N-3 position of the thymine base were prepared and evaluated for anti-HIV activity in cell culture (Lai strain of HIV-1 in CEM-c113 cells). The AZT analogs bearing a N-3 amino group (7), a hydroxyalkyl chain (12f), and a phosphonomethyl (12k) substituent displayed activities in the 0.045-0.082 microM range. The analogs 12d, 12e, 12q, 15, and 19 were active at <0.5 microM concentration. Compound 18 in which two molecules of AZT are connected at N-3 via a two-carbon link and "dimer" 11 also displayed significant activity. To obtain information concerning the mechanism of RT inhibition by these AZT analogs, compounds 7, 12d, 12e, and 12q were incubated with recombinant HIV-1 RT in the presence of poly(A)-oligo[dT(12-18)] and poly(C)-oligo[dG(12-18)] template-primers. In contrast to AZT-TP (control), none of these nucleosides displayed any significant inhibition of RT in the recombinant enzyme assay, indicating that phosphorylation is a necessary prerequisite for activity.

Synthesis and evaluation of "AZT-HEPT", "AZT-pyridinone", and "ddC-HEPT" conjugates as inhibitors of HIV reverse transcriptase.

To test the concept that HIV reverse transcriptase could be effectively inhibited by "mixed site inhibitors", a series of seven conjugates containing both a nucleoside analogue component (AZT 1, ddC 2) and a nonnucleoside type inhibitor (HEPT analogue 12, pyridinone 27) were synthesized and evaluated for their ability to block HIV replication. The (N-3 and C-5)AZT-HEPT conjugates 15, 22, and 23 displayed 2-5 microM anti-HIV activity, but they had no effect on the replication of HIV-2 or the HIV-1 strain with the Y181C mutation. The (C-5)AZT-pyridinone conjugates 34-37 were found to be inactive. In marked contrast, the ddC-HEPT molecule 26 displayed the same potency (EC(50) = 0.45 microM) against HIV-1 (wild type and the Y181C nevirapine-resistant strain) and HIV-2 in cell culture. No synergistic effect was observed for these bis-substrate inhibitors, suggesting that the two individual inhibitor components in these molecules do not bind simultaneously in their respective sites. Interestingly, however, the results indicate that the AZT-HEPT conjugates and the ddC-HEPT derivative 26 inhibit reverse transcriptase (RT) in an opposite manner. One explanation for this difference is that the former compounds interact preferentially with the hydrophobic pocket in RT, whereas 26 (after supposed triphosphorylation) inhibits RT through binding in the catalytic site.