Acute and delayed protective effects of pharmacologically induced hypothermia in an intracerebral hemorrhage stroke model of mice.

Hemorrhagic stroke, including intracerebral hemorrhage (ICH), is a devastating subtype of stroke; yet, effective clinical treatment is very limited. Accumulating evidence has shown that mild to moderate hypothermia is a promising intervention for ischemic stroke and ICH. Current physical cooling methods, however, are less efficient and often impractical for acute ICH patients. The present investigation tested pharmacologically induced hypothermia (PIH) using the second-generation neurotensin receptor (NTR) agonist HPI-201 (formerly known as ABS-201) in an adult mouse model with ICH. Acute or delayed administrations of HPI-201 (2mg/kg bolus injection followed by 2 injections of 1mg/kg, i.p.) were initiated at 1 or 24h after ICH. HPI-201 induced mild hypothermia within 30 min and body and brain temperatures were maintained at 32.7 ± 0.4°C for at least 6h without causing observable shivering. With the 1-h delayed treatment, HPI-201-induced PIH significantly reduced ICH-induced cell death and brain edema compared to saline-treated ICH animals. When HPI-201-induced hypothermia was initiated 24h after the onset of ICH, it still significantly attenuated brain edema, cell death and blood-brain barrier breakdown. HPI-201 significantly decreased the expression of matrix metallopeptidase-9 (MMP-9), reduced caspase-3 activation, and increased Bcl-2 expression in the ICH brain. Moreover, ICH mice received 1-h delayed HPI-201 treatment performed significantly better in the neurological behavior test 48 h after ICH. All together, these data suggest that systemic injection of HPI-201 is an effective hypothermic strategy that protects the brain from ICH injury with a wide therapeutic window. The protective effect of this PIH therapy is partially mediated through the alleviation of apoptosis and neurovascular damage. We suggest that pharmacological hypothermia using the newly developed neurotensin analogs is a promising therapeutic treatment for ICH.

Synthesis and analysis of potent, more lipophilic derivatives of the bradykinin B2 receptor antagonist peptide Hoe 140.

Bradykinin (BK) is an endogenous peptide that has been implicated in several pathological conditions, hence antagonists of its activity have therapeutic potential. The decapeptide Hoe 140 is currently one of the best BK antagonists, but interest remains in finding even more potent compounds. A library of Hoe 140 derivatives was synthesized that incorporated non-natural analogs of the cationic, naturally occurring amino acids arginine (Arg) and lysine (Lys). The modified amino acids were designed to form enhanced ionic interactions due to an increase in local hydrophobicity, which promotes desolvation of the cation in water. The potencies of the resulting peptides were determined by competitive binding assays in human A431 cells expressing the BK B2 receptor. Two of the peptides synthesized were equipotent to Hoe 140 (IC(50s) 2.99 and 3.36 nM) and the most potent was demonstrated as a functional antagonist in vitro by blocking BK-mediated phosphorylation of mitogen-activated protein (MAP) kinases. The new derivatives are more hydrophobic than Hoe 140 and thus may exhibit changes in pharmacokinetic properties when evaluated in vivo.

Topography of the neurotensin (NT)(8-9) binding site of human NT receptor-1 probed with NT(8-13) analogs.

A series of neurotensin (NT)(8-13) analogs featuring substitution of the Arg8 and/or Arg9 residues with non-natural cationic amino acids was synthesized and evaluated for binding to the human NT receptor-1 (hNTR-1). The modifications were designed to probe specific steric and electrostatic requirements in the N-terminal cationic region of NT(8-13) for receptor binding as a general evaluation of the feasibility of incorporating minor structural changes into a peptide at a crucial polar receptor binding site. Many of the non-natural amino acids are more or less isosteric to Arg but more lipophilic as a result of addition of alkyl groups or through removal or replacement of NH character with methylene or methyl substituents, whereas others vary the distance between the cation and the alpha-amino acid carbon. Substitution of Arg8 with N(G)-alkylated Arg derivatives or homolysine (Hlys) maintained the subnanomolar affinity of NT(8-13) to the hNTR-1. Position 8 incorporation of Hlys produced the most favorable primary amine side-chain substitution to date. Moderate losses in affinity observed with position 9 substitutions were attributed to adverse steric effects. Doubly substituted [Hlys8, DAB9]NT(8-13), in which DAB is 2,4-diaminobutyric acid, was also prepared and tested as the shorter side-chain of DAB is known to be favored in position 9 of NT(8-13). This analog maintained 60% of NT(8-13) binding affinity making it the most favored des-guanidinium-containing analog known. These results demonstrate that adequate receptor binding affinity can be maintained over a structural range of Arg analogs, thus providing a range of peptides expected to exhibit altered pharmacokinetic properties. From the standpoint of the hNTR-1 cationic binding sites, these results help to map out the structural stringency inherent in the formation of a tight binding complex with NT(8-13) and related analogs.

Discovering patterns in Plasmodium falciparum genomic DNA.

A method has been developed for discovering patterns in DNA sequences. Loosely based on the well-known Lempel Ziv model for text compression, the model detects repeated sequences in DNA. The repeats can be forward or inverted, and they need not be exact. The method is particularly useful for detecting distantly related sequences, and for finding patterns in sequences of biased nucleotide composition, where spurious patterns are often observed because the bias leads to coincidental nucleotide matches. We show here the utility of the method by applying it to genomic sequences of Plasmodium falciparum. A single scan of chromosomes 2 and 3 of P. falciparum, using our method and no other a priori information about the sequences, reveals regions of low complexity in both telomeric and central regions, long repeats in the subtelomeric regions, and shorter repeat areas in dense coding regions. Application of the method to a recently sequenced contig of chromosome 10 that has a particularly biased base composition detects a long internal repeat more readily than does the conventional dot matrix plot. Space requirements are linear, so the method can be used on large sequences. The observed repeat patterns may be related to large-scale chromosomal organization and control of gene expression. The method has general application in detecting patterns of potential interest in newly sequenced genomic material.

Selectivity enhancement induced by substitution of non-natural analogues of arginine and lysine in arginine-based thrombin inhibitors.

Seven non-natural analogues of arginine and lysine have been substituted in an established arginine-based thrombin inhibitor. Four of the new compounds exhibited significant thrombin inhibition (K(i)'s 0.53-3.95 microM) and were subsequently tested for selectivity against trypsin. The two best compounds gave selectivity ratios of 962 and 525 (trypsin/thrombin), improving upon the parent compound.

Fast, optimal alignment of three sequences using linear gap costs.

Alignment algorithms can be used to infer a relationship between sequences when the true relationship is unknown. Simple alignment algorithms use a cost function that gives a fixed cost to each possible point mutation-mismatch, deletion, insertion. These algorithms tend to find optimal alignments that have many small gaps. It is more biologically plausible to have fewer longer gaps rather than many small gaps in an alignment. To address this issue, linear gap cost algorithms are in common use for aligning biological sequence data. More reliable inferences are obtained by aligning more than two sequences at a time. The obvious dynamic programming algorithm for optimally aligning k sequences of length n runs in O(n(k)) time. This is impractical if k>/=3 and n is of any reasonable length. Thus, for this problem there are many heuristics for aligning k sequences, however, they are not guaranteed to find an optimal alignment. In this paper, we present a new algorithm guaranteed to find the optimal alignment for three sequences using linear gap costs. This gives the same results as the dynamic programming algorithm for three sequences, but typically does so much more quickly. It is particularly fast when the (three-way) edit distance is small. Our algorithm uses a speed-up technique based on Ukkonen's greedy algorithm (Ukkonen, 1983) which he presented for two sequences and simple costs.

Design rationale, synthesis, and characterization of non-natural analogs of the cationic amino acids arginine and lysine.

A series of non-natural isosteric analogs of the cationic, ion-pairing, natural amino acids arginine and lysine have been synthesized, characterized with regard to relevant physical parameters, and protected for routine inclusion in Merrifield solid-phase synthesis. The design of these molecules is based on the concept of steric inhibition of solvation, in that judicious placement of alkyl groups can destabilize aqueous ion solvation and favor ion-pairing [see Beeson & Dix (1993) J. Am. Chem. Soc. 115, 10275]. When the residues are substituted for the natural amino acids in biologically active peptides, enhanced ion-pairing of the peptides to their receptors to increase the peptides' biological activities can result. The increased lipophilicity of the non-natural residues can also improve pharmacokinetic parameters and agonist/antagonist behaviors of peptides. While the synthesis of the L-series is described, the D-isomers were also prepared using identical chemistry.

Synthesis of neurotensin(9-13) analogues exhibiting enhanced human neurotensin receptor binding affinities.

Recent evidence is consistent with neurotensin (NT)(8-13) adopting a Type I beta-turn conformation while binding the NT receptor, which would place the cationic side-chains of Arg(8) and Arg(9) in close proximity. This was the basis for the design, synthesis and analysis of truncated NT(9-13) analogues 1-5 with dicationic position 9 side-chains to emulate the functions of the 8 and 9 side-chains of NT(8-13).

Sequence complexity for biological sequence analysis.

A new statistical model for DNA considers a sequence to be a mixture of regions with little structure and regions that are approximate repeats of other subsequences, i.e. instances of repeats do not need to match each other exactly. Both forward- and reverse-complementary repeats are allowed. The model has a small number of parameters which are fitted to the data. In general there are many explanations for a given sequence and how to compute the total probability of the data given the model is shown. Computer algorithms are described for these tasks. The model can be used to compute the information content of a sequence, either in total or base by base. This amounts to looking at sequences from a data-compression point of view and it is argued that this is a good way to tackle intelligent sequence analysis in general.

Synthesis and human neurotensin receptor binding activities of neurotensin(8-13) analogues containing position 8 alpha-azido-N-alkylated derivatives of ornithine, lysine, and homolysine.

A series of neurotensin(8-13) (NT) analogues were synthesized through intermediates in which the N-terminal Arg(8) was replaced by various omega-bromo-2(S)-azido residues spanning 3-5 methylene units in side-chain length. Subsequent nucleophilic substitution of the omega-bromo groups with ammonia, methylamine, dimethylamine, or trimethylamine provided peptides containing N-terminal 2(S)-azido residues containing primary through quaternary N-alkylated side chains corresponding in length to ornithine, Lys, and homolysine. The synthetic procedure appears applicable for incorporation of a wide variety of amine-containing nonnatural amino acids into peptides. The particular modifications were intended to enhance physiochemical properties of NT(8-13) responsible for human NT 1 receptor (hNTR) binding, overall lipophilicity, and stability that may influence the potency of the peptides in vivo. When the peptides were tested for hNTR binding, the affinities in each series followed the order primary > secondary > tertiary > quaternary amine with the homolysine side-chain length being favored. All analogues possess binding affinities between acetyl-NT(8-13) and NT(8-13) indicating that the sterically less bulky alpha-azido may be inherently preferable to the acetyl group for N-terminal protection. This study extends the strategy of modifying amine-containing drugs through alkylations to peptide modification. The set of alkylated side chains also offers a new method of steric selection between receptor subtypes and could be used to modify the properties and biological effects of any peptide that contains cationic residues.

Preparation and receptor binding affinities of cyclic C-terminal neurotensin (8-13) and (9-13) analogues.

Cyclic analogues of neurotensin (NT) C-terminal fragments NT(8-13) and NT(9-13) were produced via intramolecular nucleophilic substitution of the Tyr(11) phenoxide anion on a 6-bromohexanoyl side chain substituted at position 8 or 9 and tested for NT receptor binding affinity.

Oxidation of DNA bases, deoxyribonucleosides and homopolymers by peroxyl radicals.

DNA base oxidation is considered to be a key event associated with disease initiation and progression in humans. Peroxyl radicals (ROO. ) are important oxidants found in cells whose ability to react with the DNA bases has not been characterized extensively. In this paper, the products resulting from ROO. oxidation of the DNA bases are determined by gas chromatography/MS in comparison with authentic standards. ROO. radicals oxidize adenine and guanine to their 8-hydroxy derivatives, which are considered biomarkers of hydroxyl radical (HO.) oxidations in cells. ROO. radicals also oxidize adenine to its hydroxylamine, a previously unidentified product. ROO. radicals oxidize cytosine and thymine to the monohydroxy and dihydroxy derivatives that are formed by oxidative damage in cells. Identical ROO. oxidation profiles are observed for each base when exposed as deoxyribonucleosides, monohomopolymers and base-paired dihomopolymers. These results have significance for the development, utilization and interpretation of DNA base-derived biomarkers of oxidative damage associated with disease initiation and propagation, and support the idea that the mutagenic potential of N-oxidized bases, when generated in cellular DNA, will require careful evaluation. Adenine hydroxylamine is proposed as a specific molecular probe for the activity of ROO. in cellular systems.

Compression of strings with approximate repeats.

We describe a model for strings of characters that is loosely based on the Lempel Ziv model with the addition that a repeated substring can be an approximate match to the original substring; this is close to the situation of DNA, for example. Typically there are many explanations for a given string under the model, some optimal and many suboptimal. Rather than commit to one optimal explanation, we sum the probabilities over all explanations under the model because this gives the probability of the data under the model. The model has a small number of parameters and these can be estimated from the given string by an expectation-maximization (EM) algorithm. Each iteration of the EM algorithm takes O(n2) time and a few iterations are typically sufficient. O(n2) complexity is impractical for strings of more than a few tens of thousands of characters and a faster approximation algorithm is also given. The model is further extended to include approximate reverse complementary repeats when analyzing DNA strings. Tests include the recovery of parameter estimates from known sources and applications to real DNA strings.

Discovering simple DNA sequences by compression.

An information-theoretic DNA compression scheme devised by Milosavljevic and Jurka (1993) has been used in many places in the literature for both the discovery of new genes and the compression of DNA. Their compression method applies an encoding of previously occurring runs. They use 5 different code-words: four being the DNA bases, A, C, G and T, and the other being a pointer to a previously occurring run. They advocate a code-word of length log2 5 for each of these and then encoding a run by a code-word of length 2 x log2 n, where n is the length of the sequence. This scheme encodes the start of the sequence with a code-word of length log2 n and likewise encodes the end of the sequence with a code-word of length log2 n. In this paper, we show the above coding scheme to be inefficient in various ways and improve upon it so that it can compress DNA. We discuss our implementation of various schemes some of which run in linear time.

Comparison of clone-ordering algorithms used in physical mapping.

In this paper, a number of existing and novel techniques are considered for ordering cloned extracts from the genome of an organism based on fingerprinting data. A metric is defined for comparing the quality of the clone order for each technique. Simulated annealing is used in combination with several different objective functions. Empirical results with many simulated data sets for which the correct solution is known indicate that a simple greedy algorithm with some subsequent stochastic shuffling provides the best solution. Other techniques that attempt to weight comparisons between nonadjacent clones bias the ordering and give worse results. We show that this finding is not surprising since without detailed attempts to reconcile the data into a detailed map, only approximate maps can be obtained. Making N2 pieces of data from measurements of N clones cannot improve the situation.

Redox-mediated activation of latent transforming growth factor-beta 1.

Transforming growth factor beta 1 (TGF beta) is a multifunctional cytokine that orchestrates response to injury via ubiquitous cell surface receptors. The biological activity of TGF beta is restrained by its secretion as a latent complex (LTGF beta) such that activation determines the extent of TGF beta activity during physiological and pathological events. TGF beta action has been implicated in a variety of reactive oxygen-mediated tissue processes, particularly inflammation, and in pathologies such as reperfusion injury, rheumatoid arthritis, and atherosclerosis. It was recently shown to be rapidly activated after in vivo radiation exposure, which also generates reactive oxygen species (ROS). In the present studies, the potential for redox-mediated LTGF beta activation was investigated using a cell-free system in which ROS were generated in solution by ionizing radiation or metal ion-catalyzed ascorbate reaction. Irradiation (100 Gray) of recombinant human LTGF beta in solution induced 26% activation compared with that elicited by standard thermal activation. Metal-catalyzed ascorbate oxidation elicited extremely efficient recombinant LTGF beta activation that matched or exceeded thermal activation. The efficiency of ascorbate activation depended on ascorbate concentrations and the presence of transition metal ions. We postulate that oxidation of specific amino acids in the latency-conferring peptide leads to a conformation change in the latent complex that allows release of TGF beta. Oxidative activation offers a novel route for the involvement of TGF beta in tissue processes in which ROS are implicated and endows LTGF beta with the ability to act as a sensor of oxidative stress and, by releasing TGF beta, to function as a signal for orchestrating the response of multiple cell types. LTGF beta redox sensitivity is presumably directed toward recovery of homeostasis; however, oxidation may also be a mechanism of LTGF beta activation that can be deleterious during disease mechanisms involving chronic ROS production.

Mechanism of site-selective DNA nicking by the hydrodioxyl (perhydroxyl) radical.

In previous studies, the ability of the hydrodioxyl (perhydroxyl) radical [HOO., the conjugate acid of superoxide (O2.-] to "nick" DNA under biomimetic conditions was demonstrated, and a sequence selectivity was observed. A background level of nonspecific nicking also was noted. This paper provides support for 5'-hydrogen atom abstraction from the deoxyribose ring as the initial event in the sequence-selective nicking by 02.-/HOO.. Two experiments support the proposed mechanism. First, using a defined sequence 5'-32P-labeled restriction fragment as the DNA substrate, only free (unalkylated) 3'-phosphate is produced at the site of nicking. Second, using poly (dA).poly (T) as the substrate, furfural is formed in the reaction from deoxyribose ring breakdown. Both results are consistent with 5'-hydrogen atom abstraction for initiation of the site-selective nicking. Hydrogen atom abstraction at other sites of the deoxyribose ring and/or base oxidation and loss followed by strand scission likely are responsible for the nonspecific nicking. The 5'-abstraction mechanism contrasts to those elicited by other 02-derived and metal-associated oxidants, which may provide a biomarker for the reactivity of HOO. in vivo.

Circular clustering of protein dihedral angles by Minimum Message Length.

Early work on proteins identified the existence of helices and extended sheets in protein secondary structures, a high-level classification which remains popular today. Using the Snob program for information-theoretic Minimum Message Length (MML) classification, we are able to take the protein dihedral angles as determined by X-ray crystallography, and cluster sets of dihedral angles into groups. Previous work by Hunter and States has applied a similar Bayesian classification method, AutoClass, to protein data with site position represented by 3 Cartesian co-ordinates for each of the alpha-Carbon, beta-Carbon and Nitrogen, totalling 9 co-ordinates. By using the von Mises circular distribution in the Snob program, we are instead able to represent local site properties by the two dihedral angles, phi and psi. Since each site can be modelled as having 2 degrees of freedom, this orientation-invariant dihedral angle representation of the data is more compact than that of nine highly-correlated Cartesian co-ordinates. Using the information-theoretic message length concepts discussed in the paper, such a more concise model is more likely to represent the underlying generating process from which the data came. We report on the results of our classification, plotting the classes in (phi, psi) space; and introducing a symmetric information-theoretic distance measure to build a minimum spanning tree between the classes. We also give a transition matrix between the classes and note the existence of three classes in the region phi approximately -1.09 rad and psi approximately -0.75 rad which are close on the spanning tree and have high inter-transition probabilities. This gives rise to a tight, abundant and self-perpetuating structure.

A restriction mapping engine using constraint logic programming.

Restriction mapping generally requires the application of information from various digestions by restriction enzymes to find solution sets. We use both the predicate calculus and constraint solving capabilities of CLP(R) to develop an engine for restriction mapping. Many of the techniques employed by biologists to manually find solutions are supported by the engine in a consistent manner. We provide generalized pipeline and cross-multiply operators for combining sub-maps. Our approach encourages the building of maps iteratively. We show how other techniques can be readily incorporated.

Hydrodioxyl (perhydroxyl), peroxyl, and hydroxyl radical-initiated lipid peroxidation of large unilamellar vesicles (liposomes): comparative and mechanistic studies.

The mechanisms and relative efficiencies of lipid peroxidation initiation by biological O2-derived oxidants were studied using large unilammellar vesicle (LUV) liposomes, structural models for biological membranes, as targets for oxidation. LUVs, when prepared from dilinoleoylphosphatidylcholine (DLPC) containing either 0 or 5 mol% hydroperoxide (LOOH, added either as a linoleic acid or DLPC hydroperoxide), maintained structural integrity, which enabled evaluation of the relative ability of oxidants to initiate lipid peroxidation when generated outside of the bilayer. LUVs were more oxidazable than multilamellar vesicles or lipids dispersed in solution, supporting their appropriateness as biological membrane models. In parallel to previous results using lipid dispersions (J. Aikens and T. A. Dix, 1991, J. Biol. Chem. 266, 15091-15098), both perhydroxyl (HOO.) and peroxyl (ROO.) radicals initiated lipid peroxidation in LUVs. Oxidants that did not initiate included H2O2, organic hydroperoxides, and, most notably, superoxide (O2-). HOO. and ROO. initiated by different mechanisms: HOO. required the presence of the preexisting LOOHs for efficient initiation, indicating the direct reaction of HOO. with LOOH, whereas ROO. initiated by hydrogen atom abstraction at the bisallylic site of unsaturation on the fatty acid side chain of the PCs. Hydroxyl radicals (HO.s) were poor initiators in comparison to ROO.s (and, indirectly, HOO.s), which might be considered surprising as the latter species are chemically weaker oxidants. The decreased activity of HO. was not due to decreased access to the LUVs; rather, this oxidant appears to react to generate less viable lipid peroxidation propagating species. It was also demonstrated that the fluidity of the LUV membrane had little effect on the relative initiating activity of each oxidant. It is argued that HO. may initiate lipid peroxidation only indirectly in vivo (through the generation of carbon-based peroxyl radicals, ROO.s) and that greater effort should be made to understand the roles of HOO. and ROO. at lipid peroxidation initiation.