Tauroursodeoxycholic acid binds to the G-protein site on light activated rhodopsin.

The heterotrimeric G-protein binding site on G-protein coupled receptors remains relatively unexplored regarding its potential as a new target of therapeutic intervention or as a secondary site of action by the existing drugs. Tauroursodeoxycholic acid bears structural resemblance to several compounds that were previously identified to specifically bind to the light-activated form of the visual receptor rhodopsin and to inhibit its activation of transducin. We show that TUDCA stabilizes the active form of rhodopsin, metarhodopsin II, and does not display the detergent-like effects of common amphiphilic compounds that share the cholesterol scaffold structure, such as deoxycholic acid. Computer docking of TUDCA to the model of light-activated rhodopsin revealed that it interacts using similar mode of binding to the C-terminal domain of transducin alpha subunit. The ring regions of TUDCA made hydrophobic contacts with loop 3 region of rhodopsin, while the tail of TUDCA is exposed to solvent. The results show that TUDCA interacts specifically with rhodopsin, which may contribute to its wide-ranging effects on retina physiology and as a potential therapeutic compound for retina degenerative diseases.

Sertoli cell differentiation in rhesus monkey (Macaca mulatta) is an early event in puberty and precedes attainment of the adult complement of undifferentiated spermatogonia.

In primates, the time course of Sertoli cell proliferation and differentiation during puberty and its relationship with the expansion of undifferentiated type A spermatogonia that occurs at this critical stage of development are poorly defined. Mid and late juvenile and early and late pubertal male rhesus monkeys were studied. Testes were immersion fixed, embedded in paraffin, and sectioned at 5 µm. Sertoli cell number per testis, S-phase labeling (BrdU), and growth fraction (Ki67 labeling) were determined and correlated with corresponding parameters for undifferentiated type A spermatogonia (A dark and A pale). Dual fluorescence labeling was used in addition to histochemistry to monitor spermatogonial differentiation during the peripubertal period using GFRα-1 and cKIT as markers. While the adult complement of Sertoli cells/testis was attained in early pubertal monkeys after only a few weeks of exposure to the elevated gonadotropin secretion characteristic of this developmental stage, the number of undifferentiated type A spermatogonia several months later in mid pubertal monkeys was only 50% of that in adult testes. Both A dark and A pale spermatogonia exhibited high S-phase BrdU labeling at all stages of juvenile and pubertal development. Spermatogonial differentiation, as reflected histochemically and by relative changes in GFRα-1 and cKIT expression, was not observed until after the initiation of puberty. In the rhesus monkey and maybe in other higher primates including human, the pubertal proliferation of undifferentiated spermatogonia is insidious and proceeds in the wake of a surge in Sertoli cell proliferation following termination of the juvenile stage of development.

A re-examination of proliferation and differentiation of type A spermatogonia in the adult rhesus monkey (Macaca mulatta).

BACKGROUND: Companion studies using an experimental non-human primate paradigm known as a testicular clamp indicated that the behavior of undifferentiated type A spermatogonia did not conform fully to earlier classical models. This issue was therefore re-examined in normal monkeys. METHODS: Adult male rhesus monkeys (n = 4) received an i.v. bolus of 5-bromo-2'-deoxyuridine (BrdU): one testis (first) was removed 3 h later and the remaining testis (second) was removed after 11 days and 3 h. Tissue was fixed in Bouin's solution, and numbers of A dark (Ad), small A pale (Aps) and large A pale spermatogonia, differentiating B spermatogonia, S-phase-labeled and degenerating cells were enumerated. Data are given as mean +/- SEM. RESULTS: During the early stages of the seminiferous epithelial cycle in the first testis, Ap spermatogonia (1.3 cells/cross section) were predominantly Aps (nuclear dia., 7.1 +/- 0.1 microm). Aps were never S-phase labeled. Apl (nuclear dia., 8.8 +/- 0.5 microm) appeared in Stages IV-VI and were maximal in Stages VII-X when S-phase labeling of this phenotype at 3 h was greatest. The first generation of B spermatogonia appeared in Stages XI-XII (0.84 cells/cross section). Using cells/cross section, the ratio of Ap (Stages I-V):B1:B2:B3:B4:preleptotene spermatocyte was 1:0.7:1.4:2.8:5.6:11.2. In the second testis, labeled Aps (and Apl) were observed. Ad were not BrdU labeled, and degenerating cells were rarely observed. CONCLUSIONS: The results are not entirely consistent with earlier models of spermatogonial proliferation and differentiation in the monkey. Most notably, our findings suggest that in any one cycle of the seminiferous epithelium only a fraction of Ap spermatogonia is mitotically active.

A selective monotropic elevation of FSH, but not that of LH, amplifies the proliferation and differentiation of spermatogonia in the adult rhesus monkey (Macaca mulatta).

BACKGROUND: Unilateral orchidectomy in monkeys increases spermatogenesis in the remaining testis in association with elevated follicle-stimulating hormone (FSH) secretion and testicular testosterone. The present study examined the relative importance of FSH and testosterone in driving the primate testis toward its spermatogenic ceiling. METHODS: Adult male rhesus monkeys were treated with a gonadotropin-releasing hormone receptor antagonist to inhibit endogenous FSH and luteinizing hormone (LH) secretion. The gonadotrophin drive to the testis was replaced with a pulsatile recombinant human FSH and LH infusion to maintain testicular volume and circulating testosterone and inhibin B at physiological levels. A selective monotropic elevation of FSH or LH that doubled the concentrations of inhibin B or testosterone, respectively, was then imposed for 4 weeks, each in a group of four monkeys. In a third group (n = 4), the gonadotrophin drive remained clamped at physiological levels. Bromo-deoxyuridine was administered 3 h prior to castration, and the effects of the monotropic hormone increments on germ cell number, S-phase labeling and degeneration were determined. RESULTS: Increased FSH, but not LH, produced increases in testicular volume (P < 0.05), the proportion of A pale spermatogonia entering the cell cycle and the numbers of differentiated spermatogonia and more advanced germ cells (P < 0.05). Indexes for spermatogonia labeling and germ cell degeneration were not affected. CONCLUSIONS: Under physiological conditions, circulating concentrations of FSH directly dictate sperm output of the primate testis by regulating the proportion of Ap spermatogonia in the growth fraction. An effect of FSH on survival of the first generation of differentiated B spermatogonia is not excluded.

Molecular mechanisms of constitutive activity: mutations at position 111 of the angiotensin AT1 receptor.

A possible molecular mechanism for the constitutive activity of mutants of the angiotensin type 1 receptor (AT1) at position 111 was suggested by molecular modeling. This involves a cascade of conformational changes in spatial positions of side chains along transmembrane helix (TM3) from L112 to Y113 to F117, which in turn, results in conformational changes in TM4 (residues I152 and M155) leading to the movement of TM4 as a whole. The mechanism is consistent with the available data of site-directed mutagenesis, as well as with correct predictions of constitutive activity of mutants L112F and L112C. It was also predicted that the double mutant N111G/L112A might possess basal constitutive activity comparable with that of the N111G mutant, whereas the double mutants N111G/Y113A, N111G/F117A, and N111G/I152A would have lower levels of basal activity. Experimental studies of the above double mutants showed significant constitutive activity of N111G/L112A and N111G/F117A. The basal activity of N111G/I152A was higher than expected, and that of N111G/Y113A was not determined due to poor expression of the mutant. The proposed mechanism of constitutive activity of the AT(1) receptor reveals a novel nonsimplistic view on the general problem of constitutive activity, and clearly demonstrates the inherent complexity of the process of G protein-coupled receptor (GPCR) activation.

A switch from continuous to episodic testicular testosterone release in response to pulsatile LH stimulation in juvenile rhesus monkeys (Macaca mulatta).

This study examined the ontogeny of the testicular testosterone response to precocious pulsatile LH stimulation in the juvenile rhesus monkey. LH stimulation was achieved with an i.v. infusion (one pulse every 3 h) of either single-chain human (sch)LH, administered alone or in combination with recombinant human (rh)FSH, or recombinant monkey (rm)LH in combination with rmFSH. Homologous gonadotropin treatment resulted in an adult profile of circulating mLH concentrations. The schLH infusions produced a similar pulsatile pattern in circulating LH with peak concentrations of approximately 5 IU/l. Although a robust testicular testosterone response was observed after 24 h of intermittent LH stimulation, surprisingly testosterone release at this time was continuous. The apulsatile mode of testosterone secretion, however, did not persist, and a switch to an unequivocal episodic mode of secretion, comparable to that observed in adult monkeys, occurred by day 4 of LH stimulation. FSH did not influence the pattern of the testosterone response. We conclude from these findings that progenitor Leydig cells in the primate testis are able to respond rapidly to a physiological LH stimulus. While the cell biology underlying the switch from a continuous to a pulsatile mode of testosterone secretion remains unclear, we suggest that this phenomenon may be related to the hypothesis that episodic testosterone secretion is required for the operation of the neuroendocrine axis governing testicular function.

Institutionalising cost sharing for catchment management: lessons from land and water management planning in Australia.

A recurring theme in recent Australian reports on integrated catchment management (ICM) has been the need to institutionalise more formally the cost-sharing commitments made within this domain. This represents a significant departure from earlier visions of ICM as essentially promoting voluntary uptake of resource-conservation measures. Two important questions raised by this nascent policy shift are addressed in this paper. Firstly, how might cost-sharing arrangements be given greater formality without undermining the efforts of ICM to increase the preparedness of civil stakeholders to voluntarily, or informally, accept responsibility for sharing costs? Secondly, how is it possible to formalise cost-sharing arrangements so that the transaction costs of enforcing compliance with them remain affordable? Answers to these questions are explored through a case study of the Land and Water Management Planning Program now being successfully implemented in the irrigation districts of the central-Murray region of southern inland New South Wales (NSW) surrounding Deniliquin. The sophisticated system of institutional arrangements introduced in the program to facilitate monitoring, enforcement and adaptive management of cost-sharing commitments is discussed, and insights into how informally motivated cooperation can enhance the affordability and political feasibility of formal arrangements are presented.

The functional significance of FSH in spermatogenesis and the control of its secretion in male primates.

The aim of this review is to provide an integrative analysis of the role of FSH in the control of testicular function in higher primates, including man. Attention is focused on the action of FSH during neonatal development, puberty, and adulthood. Whether FSH is the major determinant of the adult complement of Sertoli cells and whether FSH is obligatory for the initiation, maintenance, and restoration of spermatogenesis is evaluated. The mechanism whereby the circulating concentration of FSH regulates spermatogonial proliferation to dictate the sperm production rate under physiological conditions in the adult is discussed in detail. Inhibin B is the major component of the testicular negative feedback signal governing FSH beta gene expression and FSH secretion, and the evidence for this view is presented. The review concludes with the presentation of a model for the operation of the FSH-inhibin B feedback control system regulating sperm production postpubertally in monkey and man, and with speculation on issues of clinical interest.

The development and application of a novel safety-catch linker for BOC-based assembly of libraries of cyclic peptides.

Cyclic peptides are appealing targets in the drug-discovery process. Unfortunately, there currently exist no robust solid-phase strategies that allow the synthesis of large arrays of discrete cyclic peptides. Existing strategies are complicated, when synthesizing large libraries, by the extensive workup that is required to extract the cyclic product from the deprotection/cleavage mixture. To overcome this, we have developed a new safety-catch linker. The safety-catch concept described here involves the use of a protected catechol derivative in which one of the hydroxyls is masked with a benzyl group during peptide synthesis, thus making the linker deactivated to aminolysis. This masked derivative of the linker allows BOC solid-phase peptide assembly of the linear precursor. Prior to cyclization, the linker is activated and the linear peptide deprotected using conditions commonly employed (TFMSA), resulting in deprotected peptide attached to the activated form of the linker. Scavengers and deprotection adducts are removed by simple washing and filtration. Upon neutralization of the N-terminal amine, cyclization with concomitant cleavage from the resin yields the cyclic peptide in DMF solution. Workup is simple solvent removal. To exemplify this strategy, several cyclic peptides were synthesized targeted toward the somatostatin and integrin receptors. From this initial study and to show the strength of this method, we were able to synthesize a cyclic-peptide library containing over 400 members. This linker technology provides a new solid-phase avenue to access large arrays of cyclic peptides.

Rhodopsin-transducin interface: studies with conformationally constrained peptides.

To probe the interaction between transducin (G(t)) and photoactivated rhodopsin (R*), 14 analog peptides were designed and synthesized restricting the backbone of the R*-bound structure of the C-terminal 11 residues of G(t)alpha derived by transferred nuclear Overhauser effect (TrNOE) NMR. Most of the analogs were able to bind R*, supporting the TrNOE structure. Improved affinities of constrained peptides indicated that preorganization of the bound conformation is beneficial. Cys347 was found to be a recognition site; particularly, the free sulfhydryl of the side chain seems to be critical for R* binding. Leu349 was another invariable residue. Both Ile and tert-leucine (Tle) mutations for Leu349 significantly reduced the activity, indicating that the Leu side chain is in intimate contact with R*. The structure of R* was computer generated by moving helix 6 from its position in the crystal structure of ground-state rhodopsin (R) based on various experimental data. Seven feasible complexes were found when docking the TrNOE structure with R* and none with R. The analog peptides were modeled into the complexes, and their binding affinities were calculated. The predicted affinities were compared with the measured affinities to evaluate the modeled structures. Three models of the R*/G(t)alpha complex showed strong correlation to the experimental data.

Androgen depletion activates telomerase in the prostate of the nonhuman primate, Macaca mulatta.

BACKGROUND: The activity of telomerase, an enzyme that synthesizes telomeric repeats at the ends of chromosomes, is not detectable in normal human prostate. However, the majority of human prostate cancers exhibit telomerase activity. Since androgens play a major role in prostate tumorigenesis, we investigated the effect of androgen-depletion on the expression of telomerase activity in the prostate. METHODS: Adult male rhesus monkeys were either bilaterally castrated or subjected to sham surgery (n = 5 each). Approximately 6 weeks later, the animals were killed and the different regions of the prostate gland were removed and frozen immediately. Telomerase activity was assayed using the telomeric repeat amplification protocol. RESULTS: All five regions of the prostate from sham operated control animals failed to exhibit telomerase activity. In the castrated monkey, all regions of the prostate, except for the anterior lobe, expressed high levels of telomerase activity. CONCLUSIONS: Our results indicate that in monkeys, androgen-ablation leads to up-regulation of telomerase activity. The negative-regulation of telomerase activity by androgens is probably lost during prostate tumorigenesis.

3D model for TM region of the AT-1 receptor in complex with angiotensin II independently validated by site-directed mutagenesis data.

A three-dimensional model of the complex of angiotensin II (AII) with the transmembrane (TM) region of the angiotensin II receptor of type 1 (the AT-1 receptor) was obtained by molecular modeling procedures employing structural homology to the X-ray structure of rhodopsin. Since the modeling procedure considered only steric and energy considerations without prior knowledge of the experimental results of site-directed mutagenesis, the results with receptor mutants could be used for independent validation of the model. Indeed, the model brings in contact the residues of AII responsible for agonistic activity, Tyr(4), His(6), and Phe(8), with many residues of AT-1 involved in signal transduction according to site-directed mutagenesis. The model also predicts the existence of several possible conformational pathways for transferring the binding signal through the TM region of AT-1 to the intracellular loops interacting with the G-protein.

Novel approach to computer modeling of seven-helical transmembrane proteins: current progress in the test case of bacteriorhodopsin.

G-protein coupled receptors (GPCRs) are thought to be proteins with 7-membered transmembrane helical bundles (7TM proteins). Recently, the X-ray structures have been solved for two such proteins, namely for bacteriorhodopsin (BR) and rhodopsin (Rh), the latter being a GPCR. Despite similarities, the structures are different enough to suggest that 3D models for different GPCRs cannot be obtained directly employing 3D structures of BR or Rh as a unique template. The approach to computer modeling of 7TM proteins developed in this work was capable of reproducing the experimental X-ray structure of BR with great accuracy. A combination of helical packing and low-energy conformers for loops most close to the X-ray structure possesses the r.m.s.d. value of 3.13 A. Such a level of accuracy for the 3D-structure prediction for a 216-residue protein has not been achieved, so far, by any available ab initio procedure of protein folding. The approach may produce also other energetically consistent combinations of helical bundles and loop conformers, creating a variety of possible templates for 3D structures of 7TM proteins, including GPCRs. These templates may provide experimentalists with various plausible options for 3D structure of a given GPCR; in our view, only experiments will determine the final choice of the most reasonable 3D template.

Ab initio modeling of small, medium, and large loops in proteins.

This study presents different procedures for ab initio modeling of peptide loops of different sizes in proteins. Small loops (up to 8--12 residues) were generated by a straightforward procedure with subsequent "averaging" over all the low-energy conformers obtained. The averaged conformer fairly represents the entire set of low-energy conformers, root mean square deviation (RMSD) values being from 1.01 A for a 4-residue loop to 1.94 A for an 8-residue loop. Three-dimensional (3D) structures for several medium loops (20--30 residues) and for two large loops (54 and 61 residues) were predicted using residue-residue contact matrices divided into variable parts corresponding to the loops, and into a constant part corresponding to the known core of the protein. For each medium loop, a very limited number of sterically reasonable C(alpha) traces (from 1 to 3) was found; RMSD values ranged from 2.4 to 5.9 A. Single C(alpha) traces predicted for each of the large loops possessed RMSD values of 4.5 A. Generally, ab initio loop modeling presented in this work combines elements of computational procedures developed both for protein folding and for peptide conformational analysis.

Peptide interactions with G-protein coupled receptors.

Peptide recognition by G-protein coupled receptors (GPCRs) is reviewed with an emphasis on the indirect approach used to determine the receptor-bound conformation of peptide ligands. This approach was developed in response to the lack of detailed structural information available for these receptors. Recent advances in the structural determination of rhodopsin (the GPCR of the visual system) by crystallography have provided a scaffold for homology modeling of the inactive state of a wide variety of GPCRs that interact with peptide messages. Additionally, the ability to mutate GPCRs and assay compounds of similar chemical structure to test a common binding site on the receptor provides a firm experimental basis for structure-activity studies. Recognition motifs, common in other well-studied systems such as proteolytic enzymes and major histocompatibility class receptors (MHC) are reviewed briefly to provide a basis of comparison. Finally, the development of true peptidomimetics is contrasted with nonpeptide ligands, discovered through combinatorial chemistry. In many systems, the evidence suggests that the peptide ligands bind at the interface between the transmembrane segments and the extracellular loops, while nonpeptide antagonists bind within the transmembrane segments. Plausible models of GPCRs and the mechanism by which they activate G-proteins on binding peptides are beginning to emerge.

Anticonvulsant and adverse effects of avermectin analogs in mice are mediated through the gamma-aminobutyric acid(A) receptor.

Twenty-five avermectin analogs were assessed in a mouse seizure model. The ED(50) against pentylenetetrazole-induced tonic seizures ranged from 0.48 mg/kg (L-676,893) to >160 mg/kg (L-685,869) cf. 0. 26 mg/kg for diazepam. Although avermectins are without acute toxic effects, they have been historically shown to have relative low LD(50) values in mammals. The mechanisms involved in the anticonvulsant effect and the toxicity were investigated. A series of avermectin analogs displaced [(3)H]ivermectin binding to rat brain membranes and recombinant GABA(A) receptors (alpha1beta3gamma2-subtype) with the same affinities, strongly suggesting that [(3)H]ivermectin labels the GABA(A) receptor in rodent brain. Avermectins, which were anticonvulsant, were also potent inhibitors of [(3)H]ivermectin binding in rat brain. However, the rank order for anticonvulsant activity did not parallel the rank order for affinity at the [(3)H]ivermectin site and it was reasoned that avermectins may have differential affinity or efficacy at subtypes of the GABA(A) receptor. All the active compounds tested potentiated the effects of GABA at recombinant GABA(A) receptors in oocytes and at native cortical GABA(A) receptors and the efficacy of avermectins at the GABA(A) receptor correlated best with their anticonvulsant potency. Although avermectins weakly inhibited [(3)H]strychnine binding in rat spinal cord, and inhibited glycine responses on primary cultured cortical neurons, activity at glycine receptors did not correlate with either anticonvulsant activity or toxicity. Because both anticonvulsant activity and toxicity correlated best with activity at GABA(A) receptors, it is unlikely that these effects can be separated, which may contraindicate the potential use of avermectins as anticonvulsants.

Antimycobacterial pyrroles: synthesis, anti-Mycobacterium tuberculosis activity and QSAR studies.

A number of known antifungal pyrrole derivatives and some newly synthesized compounds (5-33) were tested in vitro against Mycobacterium tuberculosis CIP 103471. The majority of tested compounds were efficient antimycobacterial agents showing MIC values ranging from 0.5 to 32 microg/mL. A 3-D-QSAR study has been performed on these pyrrole derivatives to correlate their chemical structures with their observed inhibiting activity against M. tuberculosis. Due to the absence of information on a putative receptor responsible for this activity, classical quantitative structure-activity relationships (QSAR) and comparative molecular field analysis (CoMFA) have been applied. A model able to well correlate the antimycobacterial activity with the chemical structures of pyrrole derivatives 5-33 has been developed which is potentially helpful in the design of novel and more potent antituberculosis agents. The combination of CoMFA with classical QSAR descriptors led to a better hybrid 3-D-QSAR model, that successfully explains the structure-activity relationships (r2 = 0.86) of the training set. A comparison between the QSAR, CoMFA and mixed QSAR-CoMFA models is also presented. The hybrid model is to be preferred, however, because of its lowest values of the average absolute error of prediction toward a limited external test set.

Stereoselective synthesis of optically active beta-lactams, potential inhibitors of pilus assembly in pathogenic bacteria.

[reaction: see text] Optically active beta-lactams 3 are obtained in excellent yields (up to 93%) and with complete stereoselectivity from Meldrum's acid derivatives 1 and Delta(2)-thiazolines 2. A selective reduction to aldehydes 5 (R = Ar or CH(2)Ar) was then accomplished by using DIBAL-H. This rigid framework, with stereochemistry different than that of penicillin, is designed to be a suitable scaffold for the development of compounds inhibiting pilus formation in uropathogenic Escherichia coli.