Comparing protein structures: a Gaussian-based approach to the three-dimensional structural similarity of proteins.

This study describes a new method for comparing three-dimensional protein structures based on an optimal alignment of their steric fields. The method is based upon the use of spherical Gaussian functions located on individual atoms. This representation generates a flexible description of the underlying fold geometry of proteins that can be adjusted by changing the 'width' of the Gaussians. Reducing the width sharpens the representation and leads to a more 'atomlike' description; increasing the width creates a fuzzier representation that preserves the general shape features of the chain fold but with a consequent loss in atomic resolution. The width used in this study is based upon the features of individual atoms and provides a representation that is quite robust with respect to the variety of geometric features typically encountered in the alignment process. In addition, a post-alignment analysis is performed that generates sequence alignments from the corresponding structure alignments. An example, based on five mammalian and fungal matrix metalloproteinase crystal structures (human fibroblast collagenase, neutrophil collagenase, stromelysin, astacin, and adamalysin), illustrates a number of features of the Gaussian-based approach.

Identification of novel potent hydroxamic acid inhibitors of peptidyl deformylase and the importance of the hydroxamic acid functionality on inhibition.

Peptidyl deformylase (PDF) is a metallo protease that catalyzes the removal of a formyl group from the N-termini of prokaryotic prepared polypeptides, an essential step in bacterial protein synthesis. Screening of our compound collection using Staphylococcus aureus PDF afforded a very potent inhibitor with an IC(50) in the low nanomolar range. Unfortunately, the compound that contains a hydroxamic acid did not exhibit antibacterial activity (MIC). In order to address the lack of activity in the MIC assay and to determine what portion of the molecule was responsible for binding to PDF, we prepared several analogues. This paper describes our findings that the hydroxamic acid functionality found in 1 is mainly responsible for the high affinity to PDF. In addition, we identified an alternative class of PDF inhibitors, the N-hydroxy urea 18, which has both PDF and antibacterial activity.

Spontaneous activation of beta(2)- but not beta(1)-adrenoceptors expressed in cardiac myocytes from beta(1)beta(2) double knockout mice.

Although ligand-free, constitutive beta(2)-adrenergic receptor (AR) signaling has been demonstrated in naive cell lines and in transgenic mice overexpressing cardiac beta(2)-AR, it is unclear whether the dominant cardiac beta-AR subtype, beta(1)-AR, shares the ability of spontaneous activation. In the present study, we expressed human beta(1)- or beta(2)-AR via recombinant adenoviral infection in ventricular myocytes isolated from beta(1)beta(2)-AR double knockout mice, creating pure beta(1)-AR and beta(2)-AR systems with variable receptor densities. A contractile response to a nonselective beta-AR agonist, isoproterenol, was absent in double knockout mouse myocytes but was fully restored after adenoviral beta(1)-AR or adenoviral beta(2)-AR infection. Increasing the titer of adenoviral vectors (multiplicity of infection 10-1000) led to a dose-dependent expression of beta(1)- or beta(2)-AR with a maximal density of 1207 +/- 173 (36-fold over the wild-type control value) and 821+/-38 fmol/mg protein (69-fold), respectively. Using confocal immunohistochemistry, we directly visualized the cellular distribution of beta(1)-AR and beta(2)-AR and found that both subtypes were distributed on the cell surface membrane and transverse tubules, resulting in a striated pattern. In the absence of ligand, beta(2)-AR expression resulted in graded increases in baseline cAMP and contractility up to 428% and 233% of control, respectively, at the maximal beta(2)-AR density. These effects were specifically reversed by a beta(2)-AR inverse agonist, ICI 118,551 (10(-7) M). In contrast, overexpression of beta(1)-AR, even at a greater density, failed to enhance either basal cAMP or contractility; the alleged beta(1)-AR inverse agonist, CGP 20712A (10(-6) M), had no significant effect on basal contraction in these cells. Thus, we conclude that acute beta(2)-AR overexpression in cardiac myocytes elicits significant physiological responses due to spontaneous receptor activation; however, this property is beta-AR subtype specific because beta(1)-AR does not exhibit agonist-independent spontaneous activation.

A molecular-field-based similarity study of non-nucleoside HIV-1 reverse transcriptase inhibitors. 2. The relationship between alignment solutions obtained from conformationally rigid and flexible matching.

An analysis of the relationship among alignment solutions obtained from field-based matching of a representative set of rigid conformers of three non-nucleoside HIV-1 reverse transcriptase inhibitors and solutions obtained from flexible matching of the same conformers is presented. In some cases, different alignment solutions obtained from rigid matching converge to the same solution when conformational rigidity is relaxed, indicating that a reduced set of conformers per molecule may be sufficient in many field-based similarity studies. Furthermore, the results also indicate the importance of going beyond the pairwise similarity level to obtain consistent solutions in flexible-matching studies. In this respect, the best conformationally flexible multi-molecule alignment obtained is found to be in good agreement with the relative binding geometry and orientation found experimentally from protein-ligand crystal structures. The rms separation between corresponding atoms in computed and 'experimental' sets of three inhibitor structures is 0.94 A.

Efficacy of a green tea extract rich in catechin polyphenols and caffeine in increasing 24-h energy expenditure and fat oxidation in humans.

BACKGROUND: Current interest in the role of functional foods in weight control has focused on plant ingredients capable of interfering with the sympathoadrenal system. OBJECTIVE: We investigated whether a green tea extract, by virtue of its high content of caffeine and catechin polyphenols, could increase 24-h energy expenditure (EE) and fat oxidation in humans. DESIGN: Twenty-four-hour EE, the respiratory quotient (RQ), and the urinary excretion of nitrogen and catecholamines were measured in a respiratory chamber in 10 healthy men. On 3 separate occasions, subjects were randomly assigned among 3 treatments: green tea extract (50 mg caffeine and 90 mg epigallocatechin gallate), caffeine (50 mg), and placebo, which they ingested at breakfast, lunch, and dinner. RESULTS: Relative to placebo, treatment with the green tea extract resulted in a significant increase in 24-h EE (4%; P < 0.01) and a significant decrease in 24-h RQ (from 0.88 to 0.85; P < 0.001) without any change in urinary nitrogen. Twenty-four-hour urinary norepinephrine excretion was higher during treatment with the green tea extract than with the placebo (40%, P < 0.05). Treatment with caffeine in amounts equivalent to those found in the green tea extract had no effect on EE and RQ nor on urinary nitrogen or catecholamines. CONCLUSIONS: Green tea has thermogenic properties and promotes fat oxidation beyond that explained by its caffeine content per se. The green tea extract may play a role in the control of body composition via sympathetic activation of thermogenesis, fat oxidation, or both.

ERK plays a regulatory role in induction of LTP by theta frequency stimulation and its modulation by beta-adrenergic receptors.

MAP kinase (ERK) translates cell surface signals into alterations in transcription. We have found that ERK also regulates hippocampal neuronal excitability during 5 Hz stimulation and thereby regulates forms of long-term potentiation (LTP) that do not require macromolecular synthesis. Moreover, ERK-mediated changes in excitability are selectively required for some forms of LTP but not others. ERK is required for the early phase of LTP elicited by brief 5 Hz stimulation, as well as for LTP elicited by more prolonged 5 Hz stimulation when paired with beta1-adrenergic receptor activation. By contrast, ERK plays no role in LTP elicited by a single 1 s 100 Hz train. Consistent with these results, we find that ERK is activated by beta-adrenergic receptors in CA1 pyramidal cell somas and dendrites.

Promoter-activated expression of nerve growth factor for treatment of neurodegenerative diseases.

Genetic transfer approaches have received recent consideration as potential treatment modalities for human central and peripheral nervous system (CNS and PNS, respectively) neurodegenerative disorders, including Parkinson's disease, Alzheimer's disease, and amyotrophic lateral sclerosis. Transplantation of genetically modified cells into the brain represents a promising strategy for the delivery and expression of specific neurotrophic factors, neurotransmitter-synthesizing enzymes, and cellular regulatory proteins for intervention in neurodegenerative diseases. The use of specific regulatable promoters may also provide potential control of gene expression required for dose-specific or time-specific therapeutic strategies. In this article, we review the potential use of activated promoters in ex vivo systems for the potential genetic therapy of neurodegenerative disorders, and then describe our own studies using the zinc-inducible metallothionein promoter for the regulated expression of nerve growth factor (NGF) in rodent brain transplants.

The disparate effects of Alzheimer's disease and Huntington's disease on semantic memory.

Alzheimer's disease (AD) and Huntington's disease (HD) impair performance on semantic memory tasks, but researchers disagree on whether AD and HD cause these impairments in the same manner. According to one view, AD disrupts the storage of semantic memories, whereas HD disrupts the retrieval of semantic memories. Dissenters argue that AD, like HD, disrupts retrieval. In this study, participants generated category exemplars (e.g., kinds of fruits) for 1 min, and response latencies were examined. Relative to healthy controls, the 12 AD patients produced a larger proportion of responses earlier in the recall period, consistent with the view that AD patients quickly exhaust their limited supply of items in storage. By contrast, the 12 HD patients produced a larger proportion of their responses late in the recall period, consistent with the view that HD slows retrieval.

Targeted disruption of the beta2 adrenergic receptor gene.

beta-Adrenergic receptors (beta-ARs) are members of the superfamily of G-protein-coupled receptors that mediate the effects of catecholamines in the sympathetic nervous system. Three distinct beta-AR subtypes have been identified (beta1-AR, beta2-AR, and beta3-AR). In order to define further the role of the different beta-AR subtypes, we have used gene targeting to inactivate selectively the beta2-AR gene in mice. Based on intercrosses of heterozygous knockout (beta2-AR +/-) mice, there is no prenatal lethality associated with this mutation. Adult knockout mice (beta2-AR -/-) appear grossly normal and are fertile. Their resting heart rate and blood pressure are normal, and they have a normal chronotropic response to the beta-AR agonist isoproterenol. The hypotensive response to isoproterenol, however, is significantly blunted compared with wild type mice. Despite this defect in vasodilation, beta2-AR -/- mice can still exercise normally and actually have a greater total exercise capacity than wild type mice. At comparable workloads, beta2-AR -/- mice had a lower respiratory exchange ratio than wild type mice suggesting a difference in energy metabolism. beta2-AR -/- mice become hypertensive during exercise and exhibit a greater hypertensive response to epinephrine compared with wild type mice. In summary, the primary physiologic consequences of the beta2-AR gene disruption are observed only during the stress of exercise and are the result of alterations in both vascular tone and energy metabolism.

Cardiovascular and metabolic alterations in mice lacking both beta1- and beta2-adrenergic receptors.

The activation state of beta-adrenergic receptors (beta-ARs) in vivo is an important determinant of hemodynamic status, cardiac performance, and metabolic rate. In order to achieve homeostasis in vivo, the cellular signals generated by beta-AR activation are integrated with signals from a number of other distinct receptors and signaling pathways. We have utilized genetic knockout models to test directly the role of beta1- and/or beta2-AR expression on these homeostatic control mechanisms. Despite total absence of beta1- and beta2-ARs, the predominant cardiovascular beta-adrenergic subtypes, basal heart rate, blood pressure, and metabolic rate do not differ from wild type controls. However, stimulation of beta-AR function by beta-AR agonists or exercise reveals significant impairments in chronotropic range, vascular reactivity, and metabolic rate. Surprisingly, the blunted chronotropic and metabolic response to exercise seen in beta1/beta2-AR double knockouts fails to impact maximal exercise capacity. Integrating the results from single beta1- and beta2-AR knockouts as well as the beta1-/beta2-AR double knock-out suggest that in the mouse, beta-AR stimulation of cardiac inotropy and chronotropy is mediated almost exclusively by the beta1-AR, whereas vascular relaxation and metabolic rate are controlled by all three beta-ARs (beta1-, beta2-, and beta3-AR). Compensatory alterations in cardiac muscarinic receptor density and vascular beta3-AR responsiveness are also observed in beta1-/beta2-AR double knockouts. In addition to its ability to define beta-AR subtype-specific functions, this genetic approach is also useful in identifying adaptive alterations that serve to maintain critical physiological setpoints such as heart rate, blood pressure, and metabolic rate when cellular signaling mechanisms are perturbed.

Field-based similarity forcing in energy minimization and molecular matching.

A new field-based similarity forcing procedure for matching conformationally-flexible molecules is presented. The method extends earlier work on similarity matching of molecules based upon the program MIMIC, by directly coupling a similarity function to a molecular mechanics force field. In this way conformational energetics are fully accounted for in the similarity matching process. Simultaneous similarity/conformational searches can then be undertaken within a Monte Carlo or molecular dynamics framework. Here, a Monte Carlo approach is used to provide a simple example of two HIV-1 reverse transcriptase inhibitors, nevirapine and alpha APA, that illustrates the basic characteristics of the method and suggests areas for further investigation.

A molecular-field-based similarity study of non-nucleoside HIV-1 reverse transcriptase inhibitors.

This article describes a molecular-field-based similarity method for aligning molecules by matching their steric and electrostatic fields and an application of the method to the alignment of three structurally diverse non-nucleoside HIV-1 reverse transcriptase inhibitors. A brief description of the method, as implemented in the program MIMIC, is presented, including a discussion of pairwise and multi-molecule similarity-based matching. The application provides an example that illustrates how relative binding orientations of molecules can be determined in the absence of detailed structural information on their target protein. In the particular system studied here, availability of the X-ray crystal structures of the respective ligand-protein complexes provides a means for constructing an 'experimental model' of the relative binding orientations of the three inhibitors. The experimental model is derived by using MIMIC to align the steric fields of the three protein P66 subunit main chains, producing an overlay with a 1.41 A average rms distance between the corresponding C alpha's in the three chains. The inter-chain residue similarities for the backbone structures show that the main-chain conformations are conserved in the region of the inhibitor-binding site, with the major deviations located primarily in the 'finger' and RNase H regions. The resulting inhibitor structure overlay provides an experimental-based model that can be used to evaluate the quality of the direct a priori inhibitor alignment obtained using MIMIC. It is found that the 'best' pairwise alignments do not always correspond to the experimental model alignments. Therefore, simply combining the best pairwise alignments will not necessarily produce the optimal multi-molecule alignment. However, the best simultaneous three-molecule alignment was found to reproduce the experimental inhibitor alignment model. A pairwise consistency index has been derived which gauges the quality of combining the pairwise alignments and aids in efficiently forming the optimal multi-molecule alignment analysis. Two post-alignment procedures are described that provide information on feature-based and field-based pharmacophoric patterns. The former corresponds to traditional pharmacophore models and is derived from the contribution of individual atoms to the total similarity. The latter is based on molecular regions rather than atoms and is constructed by computing the percent contribution to the similarity of individual points in a regular lattice surrounding the molecules, which when contoured and colored visually depict regions of highly conserved similarity. A discussion of how the information provided by each of the procedures is useful in drug design is also presented.

Use of decompressive craniectomy after severe head trauma.

Ten percent of head injuries quality as "severe," requiring prompt diagnosis and treatment. This level I trauma center uses an aggressive approach to treatment of severe head injuries and, when indicated, intervenes with decompressive craniectomy. This article explains decompressive craniectomy, including indications, procedures, and protocols. The case studies presented highlight two patients with profound head injuries. Additionally, cranioplasty replacement of autograft flap is explained and discussed.

Physiological consequences of beta-adrenergic receptor disruption.

Activation of beta-adrenergic receptors (beta-ARs) in vivo is an important means by which animals regulate cardiac performance, vascular tone, lipid and carbohydrate metabolism, and behavior. The advent of targeted gene disruption in mice has led to significant advances in our understanding of the role that beta-AR subtypes play in these processes, and this technique has become an important tool for the study of G protein coupled receptors in general. To date, targeted disruption of both beta1- and beta3-ARs in mice has been reported. Mice lacking beta1-ARs are unresponsive to cardiac beta-AR stimulation, suggesting that neither beta2- nor beta3-ARs couple to inotropic or chronotropic responses in the mouse. Conversely, mice lacking beta3-ARs retain at least some adipose beta-AR responsiveness through remaining beta1- and beta2-ARs, suggesting that all three beta-AR subtypes mediate similar functions in this tissue. While these knockout models have been extremely valuable tools for revealing the roles that individual beta-ARs play in whole animal physiology, it is also useful to integrate the results of experiments derived from either transgenic overexpression of beta-ARs or purely pharmacological approaches to the study of beta-AR function in order to create a comprehensive model of beta-AR function in vivo.

When two memories can and cannot be retrieved concurrently.

We examined whether two memories can be retrieved concurrently from long-term memory. In Experiment 1, the subjects recalled words, either from two categories--alternating between the two--or from just one category. In Experiment 2, the subjects recalled two words belonging to either the same category or different categories, and the category prompts for these two responses appeared either simultaneously or successively. The results of both studies are consistent with the view that two items from different categories must be retrieved serially, whereas two items from the same category can be retrieved in parallel.

Insights from in vivo modification of adrenergic receptor gene expression.

Adrenergic receptors are key targets within the autonomic nervous system, regulating a wide variety of physiological processes. The ability to modify adrenergic receptor expression patterns in vivo has added a powerful new tool to the functional analysis of these receptors. Modification of adrenergic receptor gene expression by overexpression, genetic ablation, or site-specific mutation has added new insight to models of receptor coupling behavior, pharmacology, and subtype-specific physiological function. This review highlights some of the recent advances resulting from such genetic approaches to the study of adrenergic receptors.

Alterations in dynamic heart rate control in the beta 1-adrenergic receptor knockout mouse.

beta 1-Adrenergic receptors (beta 1-ARs) are key targets of sympathetic nervous system activity and play a major role in the beat-to-beat regulation of cardiac chronotropy and inotropy. We employed a beta 1-AR gene knockout model to test the hypothesis that beta 1-AR function is critical for maintenance of resting heart rate and baroreflex responsiveness and, on the basis of its important role in regulating chronotropy and inotropy, is also required for maximal exercise capacity. Using an awake unrestrained mouse model, we demonstrate that resting heart rate and blood pressure are normal in beta 1-AR knockouts and that the qualitative responses to baroreflex stimulation are intact. Chronotropic reserve in beta 1-AR knockouts is markedly limited, with peak heart rates approximately 200 beats/min less than wild types. During graded treadmill exercise, heart rate is significantly depressed in beta 1-AR knockouts at all work loads, but despite this limitation, there are no reductions in maximal exercise capacity or metabolic indexes. Thus, in mice, the beta 1-AR is not essential for either maintenance of resting heart rate or for maximally stressed cardiovascular performance.

G protein-coupled receptors: functional and mechanistic insights through altered gene expression.

G protein-coupled receptors (GPCRs) comprise a large and diverse family of molecules that play essential roles in signal transduction. In addition to a constantly expanding pharmacological repertoire, recent advances in the ability to manipulate GPCR expression in vivo have provided another valuable approach in the study of GPCR function and mechanism of action. Current technologies now allow investigators to manipulate GPCR expression in a variety of ways. Graded reductions in GPCR expression can be achieved through antisense strategies or total gene ablation or replacement can be achieved through gene targeting strategies, and exogenous expression of wild-type or mutant GPCR isoforms can be accomplished with transgenic technologies. Both the techniques used to achieve these specific alterations and the consequences of altered expression patterns are reviewed here and discussed in the context of GPCR function and mechanism of action.

Synthesis and antibacterial activity of [6,5,5] and [6,6,5] tricyclic fused oxazolidinones.

A series of conformationally restricted, [6,5,5] and [6,6,5] tricyclic fused oxazolidinones were synthesized and tested for antibacterial activity. Several compounds in the trans-[6,5,5] series demonstrated potent in vitro and in vivo activity. This work provides valuable information regarding the preferred conformational orientation of the oxazolidinones at the binding site.