The design, computer modeling, solution structure, and biological evaluation of synthetic analogs of bryostatin 1

The bryostatins are a unique family of emerging cancer chemotherapeutic candidates isolated from marine bryozoa. Although the biochemical basis for their therapeutic activity is not known, these macrolactones exhibit high affinities for protein kinase C (PKC) isozymes, compete for the phorbol ester binding site on PKC, and stimulate kinase activity in vitro and in vivo. Unlike the phorbol esters, they are not first-stage tumor promoters. The design, computer modeling, NMR solution structure, PKC binding, and functional assays of a unique class of synthetic bryostatin analogs are described. These analogs (7b, 7c, and 8) retain the putative recognition domain of the bryostatins but are simplified through deletions and modifications in the C4-C14 spacer domain. Computer modeling of an analog prototype (7a) indicates that it exists preferentially in two distinct conformational classes, one in close agreement with the crystal structure of bryostatin 1. The solution structure of synthetic analog 7c was determined by NMR spectroscopy and found to be very similar to the previously reported structures of bryostatins 1 and 10. Analogs 7b, 7c, and 8 bound strongly to PKC isozymes with Ki = 297, 3.4, and 8.3 nM, respectively. Control 7d, like the corresponding bryostatin derivative, exhibited weak PKC affinity, as did the derivative, 9, lacking the spacer domain. Like bryostatin, acetal 7c exhibited significant levels of in vitro growth inhibitory activity (1.8–170 ng/ml) against several human cancer cell lines, providing an important step toward the development of simplified, synthetically accessible analogs of the bryostatins.

Solution structure of the 2-amino-1- methyl-6-phenylimidazo[4,5-b]pyridine C8-deoxyguanosine adduct in duplex DNA

The carcinogenic heterocyclic amine (HA) 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is formed during the cooking of various meats. To enable structure/activity studies aimed at understanding how DNA damaged by a member of the HA class of compounds can ultimately lead to cancer, we have determined the first solution structure of an 11-mer duplex containing the C8-dG adduct formed by reaction with N-acetoxy-PhIP. A slow conformational exchange is observed in which the PhIP ligand either intercalates into the DNA helix by denaturing and displacing the modified base pair (main form) or is located outside the helix in a minimally perturbed B-DNA duplex (minor form). In the main base-displaced intercalation structure, the minor groove is widened, and the major groove is compressed at the lesion site because of the location of the bulky PhIP-N-methyl and phenyl ring in the minor groove; this distortion causes significant bending of the helix. The PhIP phenyl ring interacts with the phosphodiester-sugar ring backbone of the complementary strand and its fast rotation with respect to the intercalated imidazopyridine ring causes substantial distortions at this site, such as unwinding and bulging-out of the strand. The glycosidic torsion angle of the [PhIP]dG residue is syn, and the displaced guanine base is directed toward the 3′ end of the modified strand. This study contributes, to our knowledge, the first structural information on the biologically relevant HA class to a growing body of knowledge about how conformational similarities and differences for a variety of types of lesions can influence protein interactions and ultimately biological outcome.

The solution structure of the Raf-1 cysteine-rich domain: a novel ras and phospholipid binding site.

The Raf-1 protein kinase is the best-characterized downstream effector of activated Ras. Interaction with Ras leads to Raf-1 activation and results in transduction of cell growth and differentiation signals. The details of Raf-1 activation are unclear, but our characterization of a second Ras-binding site in the cysteine-rich domain (CRD) and the involvement of both Ras-binding sites in effective Raf-1-mediated transformation provides insight into the molecular aspects and consequences of Ras-Raf interactions. The Raf-1 CRD is a member of an emerging family of domains, many of which are found within signal transducing proteins. Several contain binding sites for diacylglycerol (or phorbol esters) and phosphatidylserine and are believed to play a role in membrane translocation and enzyme activation. The CRD from Raf-1 does not bind diacylglycerol but interacts with Ras and phosphatidylserine. To investigate the ligand-binding specificities associated with CRDs, we have determined the solution structure of the Raf-1 CRD using heteronuclear multidimensional NMR. We show that there are differences between this structure and the structures of two related domains from protein kinase C (PKC). The differences are confined to regions of the CRDs involved in binding phorbol ester in the PKC domains. Since phosphatidylserine is a common ligand, we expect its binding site to be located in regions where the structures of the Raf-1 and PKC domains are similar. The structure of the Raf-1 CRD represents an example of this family of domains that does not bind diacylglycerol and provides a framework for investigating its interactions with other molecules.

Determining RNA solution structure by segmental isotopic labeling and NMR: application to Caenorhabditis elegans spliced leader RNA 1.

Recent developments in multidimensional heteronuclear NMR spectroscopy and large-scale synthesis of uniformly 13C- and 15N-labeled oligonucleotides have greatly improved the prospects for determination of the solution structure of RNA. However, there are circumstances in which it may be advantageous to label only a segment of the entire RNA chain. For example, in a larger RNA molecule the structural question of interest may reside in a localized domain. Labeling only the corresponding nucleotides simplifies the spectrum and resonance assignments because one can filter proton spectra for coupling to 13C and 15N. Another example is in resolving alternative secondary structure models that are indistinguishable in imino proton connectivities. Here we report a general method for enzymatic synthesis of quantities of segmentally labeled RNA molecules required for NMR spectroscopy. We use the method to distinguish definitively two competing secondary structure models for the 5' half of Caenorhabditis elegans spliced leader RNA by comparison of the two-dimensional [15N] 1H heteronuclear multiple quantum correlation spectrum of the uniformly labeled sample with that of a segmentally labeled sample. The method requires relatively small samples; solutions in the 200-300 microM concentration range, with a total of 30 nmol or approximately 40 micrograms of RNA in approximately 150 microliters, give strong NMR signals in a short accumulation time. The method can be adapted to label an internal segment of a larger RNA chain for study of localized structural problems. This definitive approach provides an alternative to the more common enzymatic and chemical footprinting methods for determination of RNA secondary structure.

Effects of various aging heat treatments and solution-annealing and aging heat treatments on tensile, creep, and stress-rupture strengths of inconel x sheet to temperatures of 1,400 degrees F /

Cover title.

Enzymatic cyclization of a potent Bowman-Birk protease inhibitor, sunflower trypsin inhibitor-1, and solution structure of an acyclic precursor peptide

The most potent known naturally occurring Bowman-Birk inhibitor, sunflower trypsin inhibitor-1 (SFTI-1), is a bicyclic 14-amino acid peptide from sunflower seeds comprising one disulfide bond and a cyclic backbone. At present, little is known about the cyclization mechanism of SFTI-1. We show here that an acyclic permutant of SFTI-1 open at its scissile bond, SFTI-1[ 6,5], also functions as an inhibitor of trypsin and that it can be enzymatically backbone-cyclized by incubation with bovine beta-trypsin. The resulting ratio of cyclic SFTI-1 to SFTI1[6,5] is similar to9:1 regardless of whether trypsin is incubated with SFTI-1[ 6,5] or SFTI-1. Enzymatic resynthesis of the scissile bond to form cyclic SFTI-1 is a novel mechanism of cyclization of SFTI-1[ 6,5]. Such a reaction could potentially occur on a trypsin affinity column as used in the original isolation procedure of SFTI-1. We therefore extracted SFTI-1 from sunflower seeds without a trypsin purification step and confirmed that the backbone of SFTI-1 is indeed naturally cyclic. Structural studies on SFTI-1[ 6,5] revealed high heterogeneity, and multiple species of SFTI-1[ 6,5] were identified. The main species closely resembles the structure of cyclic SFTI-1 with the broken binding loop able to rotate between a cis/trans geometry of the I7-P8 bond with the cis conformer being similar to the canonical binding loop conformation. The non-reactive loop adopts a beta-hairpin structure as in cyclic wild-type SFTI-1. Another species exhibits an isoaspartate residue at position 14 and provides implications for possible in vivo cyclization mechanisms.

Solution of the dual reflection equation for A(n-1)((1)) solid-on-solid model

We obtain a diagonal solution of the dual reflection equation for the elliptic A(n-1)((1)) solid-on-solid model. The isomorphism between the solutions of the reflection equation and its dual is studied. (C) 2004 American Institute of Physics.

Isolation and characterization of novel cyclotides from Viola hederaceae - Solution structure and anti-HIV activity of vhl-1, a leaf-specific expressed cyclotide

Based on a newly established sequencing strategy featured by its efficiency, simplicity, and easy manipulation, the sequences of four novel cyclotides (macrocyclic knotted proteins) isolated from an Australian plant Viola hederaceae were determined. The three-dimensional solution structure of V. hederaceae leaf cyclotide-1 ( vhl-1), a leaf-specific expressed 31-residue cyclotide, has been determined using two-dimensional H-1 NMR spectroscopy. vhl-1 adopts a compact and well defined structure including a distorted triple-stranded β- sheet, a short 310 helical segment and several turns. It is stabilized by three disulfide bonds, which, together with backbone segments, form a cyclic cystine knot motif. The three-disulfide bonds are almost completely buried into the protein core, and the six cysteines contribute only 3.8% to the molecular surface. A pH titration experiment revealed that the folding of vhl-1 shows little pH dependence and allowed the pK(a) of 3.0 for Glu(3) and ∼ 5.0 for Glu(14) to be determined. Met(7) was found to be oxidized in the native form, consistent with the fact that its side chain protrudes into the solvent, occupying 7.5% of the molecular surface. vhl-1 shows anti-HIV activity with an EC50 value of 0.87 μ m.

Synthetic polymers for ophthalmic applications

The contact lens represents a well-established important class of biomaterials. This thesis brings together the literature, mostly Japanese and American patents, concerned with an important group of polymers, `rigid gas permeable contact lens materials'. A comparison is made of similarities in the underlying chemical themes, centring on the use of variants of highly branched siloxy compounds with polymerizable methacrylate groups. There is a need for standard techniques to assess laboratory behaviour in relation to in vitro performance. A major part of the present work is dedicated to the establishment of such standardised techniques. It is apparent that property design requirements in this field (i.e. oxygen permeability, surface and mechanical properties) are to some extent conflicting. In principle, the structural approaches used to obtain high oxygen permeability lead to surface properties that are less than ideal in terms of compatibility with tears. PMMA is known to have uniquely good (but not perfect) surface properties in this respect; it has been used as a starting point in attempting to design new materials that possess a more acceptable compromise of transport and surface properties for ocular use. Initial examination of the oxygen permeabilities of relatively simple alkyl methacrylates, show that butyl methacrylate which has a permeability some fifty times greater than PMMA, represents an interesting and hitherto unexplored group of materials for ophthalmic applications. Consideration was similarly given to surface modification techniques that would produce materials having the ability to sustain coherent tear film in the eye without markedly impairing oxygen transport properties. Particular attention is paid to the use of oxygen plasma techniques in this respect. In conclusion, similar design considerations were applied to an extended wear hydrogel lens material in an attempt to overcome mechanical stability deficiencies which manifest themselves lq`in vivo' but not `in vitro'. A relatively simple structure modification, involving steric shielding of the amide substituent group, proved to be an effective solution to the problem.

Comparative performance of analytical techniques for polyhexamethylene biguanide (PHMB) in ophthalmic solutions

Purpose: The use of PHMB as a disinfectant in contact lens multipurpose solutions has been at the centre of much debate in recent times, particularly in relation to the issue of solution induced corneal staining. Clinical studies have been carried out which suggest different effects with individual contact lens materials used in combination with specific PHMB containing care regimes. There does not appear to be, however, a reliable analytical technique that would detect and quantify with any degree of accuracy the specific levels of PHMB that are taken up and released from individual solutions by the various contact lens materials. Methods: PHMB is a mixture of positively charged polymer units of varying molecular weight that has maximum absorbance wavelength of 236 nm. On the basis of these properties a range of assays including capillary electrophoresis, HPLC, a nickelnioxime colorimetric technique, mass spectrophotometry, UV spectroscopy and ion chromatography were assessed paying particular attention to each of their constraints and detection levels. Particular interest was focused on the relative advantage of contactless conductivity compared to UV and mass spectrometry detection in capillary electrophoresis (CE). This study provides an overview of the comparative performance of these techniques. Results: The UV absorbance of PHMB solutions, ranging from 0.0625 to 50 ppm was measured at 236 nm. Within this range the calibration curve appears to be linear however, absorption values below 1 ppm (0.0001%) were extremely difficult to reproduce. The concentration of PHMB in solutions is in the range of 0.0002–0.00005% and our investigations suggest that levels of PHMB below 0.0001% (levels encountered in uptake and release studies) can not be accurately estimated, in particular when analysing complex lens care solutions which can contain competitively absorbing, and thus interfering, species in the solution. The use of separative methodologies, such as CE using UV detection alone is similarly limited. Alternative techniques including contactless conductivity detection offer greater discrimination in complex solutions together with the opportunity for dual channel detection. Preliminary results achieved by TraceDec1 contactless conductivity detection, (Gain 150%, Offset 150) in conjunction with the Agilent capillary electrophoresis system using a bare fused silica capillary (extended light path, 50 mid, total length 64.5 cm, effective length 56 cm) and a cationic buffer at pH 3.2, exhibit great potential with reproducible PHMB split peaks. Conclusions: PHMB-based solutions are commonly associated with the potential to invoke corneal staining in combination with certain contact lens materials. However this terminology ‘PHMBbased solution’ is used primarily because PHMB itself has yet to be adequately implicated as the causative agent of the staining and compromised corneal cell integrity. The lack of well characterised adequately sensitive assays, coupled with the range of additional components that characterise individual care solutions pose a major barrier to the investigation of PHMB interactions in the lenswearing eye.

Ophthalmic engineering:the development of novel instrumentation to further research in the field

The principle theme of this thesis is the advancement and expansion of ophthalmic research via the collaboration between professional Engineers and professional Optometrists. The aim has been to develop new and novel approaches and solutions to contemporary problems in the field. The work is sub divided into three areas of investigation; 1) High technology systems, 2) Modification of current systems to increase functionality, and 3) Development of smaller more portable and cost effective systems. High Technology Systems: A novel high speed Optical Coherence Tomography (OCT) system with integrated simultaneous high speed photography was developed achieving better operational speed than is currently available commercially. The mechanical design of the system featured a novel 8 axis alignment system. A full set of capture, analysis, and post processing software was developed providing custom analysis systems for ophthalmic OCT imaging, expanding the current capabilities of the technology. A large clinical trial was undertaken to test the dynamics of contact lens edge interaction with the cornea in-vivo. The interaction between lens edge design, lens base curvature, post insertion times and edge positions was investigated. A novel method for correction of optical distortion when assessing lens indentation was also demonstrated. Modification of Current Systems: A commercial autorefractor, the WAM-5500, was modified with the addition of extra hardware and a custom software and firmware solution to produce a system that was capable of measuring dynamic accommodative response to various stimuli in real time. A novel software package to control the data capture process was developed allowing real time monitoring of data by the practitioner, adding considerable functionality of the instrument further to the standard system. The device was used to assess the accommodative response differences between subjects who had worn UV blocking contact lens for 5 years, verses a control group that had not worn UV blocking lenses. While the standard static measurement of accommodation showed no differences between the two groups, it was determined that the UV blocking group did show better accommodative rise and fall times (faster), thus demonstrating the benefits of the modification of this commercially available instrumentation. Portable and Cost effective Systems: A new instrument was developed to expand the capability of the now defunct Keeler Tearscope. A device was developed that provided a similar capability in allowing observation of the reflected mires from the tear film surface, but with the added advantage of being able to record the observations. The device was tested comparatively with the tearscope and other tear film break-up techniques, demonstrating its potential. In Conclusion: This work has successfully demonstrated the advantages of interdisciplinary research between engineering and ophthalmic research has provided new and novel instrumented solutions as well as having added to the sum of scientific understanding in the ophthalmic field.

Equilibrium Sorption Studies of Hg (II) Ions from Aqueous Solution using Powdered Swamp Arum ( Lasimorpha senegalensis ) Seeds.

The potential of swamp arum ( Lasimorpha senegalensis ) seeds as a low-cost adsorbent for the removal of Hg (II) ions from aqueous solution was investigated in this study. The influence of initial metal concentration on the percent adsorption of Hg (II) ions onto powdered swamp arum seeds was studied in a batch system and the filtrate was analyzed using Atomic Absorption Spectrometry (AAS). The percent adsorbed for 10, 20, 40, 60 and 80 mg/L of the aqueous solution were 97.7, 98.9, 99.3, 99.7, and 96.5% respectively. Three isotherms; Langmuir, Freundlich, and BET were used to model the equilibrium sorption of Hg (II) ions onto powdered swamp arum seeds, with a correlation coefficient of 0.998, 0.784 and0.842 respectively. The Langmuir model fitted the equilibrium data best, with a correlation coefficient of 0.998 and a maximum adsorption capacity qm, of 5.917 mg/g. Thus, indicating monolayer coverage on the adsorbent. The results showed that swamp arum seed have the potential to be applied as alternative lowcost biosorbent in the remediation of heavy metal contamination in waste water.