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Z Lai

Publications and source records attributed to Z Lai.

At least 55 records · Page 3Linked to original sources
PubMed1997

Quantitative comparison of mu opioid receptor mRNA in selected CNS regions of alcohol naive rats selectively bred for high and low alcohol drinking.

We combined solution hybridization, ribonuclease protection and microdissection techniques to quantitatively compare the anatomical distribution of mu receptor mRNA in discrete brain regions of alcohol naive rats selectively bred for high and low alcohol drinking (HAD and LAD lines, respectively). The solution hybridization assay is highly sensitive and can detect mu opioid receptor mRNA in a 100-fold linear range from 4 to 421 amol. HAD and LAD rats exhibited a similar level of mu receptor mRNA in all central nervous system (CNS) regions examined except for the inferior colliculus. Our data suggest that the steady-state level of mu receptor mRNA is not associated with genetic differences in alcohol drinking behavior.

Alcohol Drinking↗
PubMed1997

Altered hematopoiesis, behavior, and sexual function in mu opioid receptor-deficient mice.

The mu opioid receptor is thought to be the cellular target of opioid narcotics such as morphine and heroin, mediating their effects in both pain relief and euphoria. Its involvement is also implicated in a range of diverse biological processes. Using a mouse model in which the receptor gene was disrupted by targeted homologous recombination, we explored the involvement of this receptor in a number of physiological functions. Mice homozygous for the disrupted gene developed normally, but their motor function was altered. Drug-naive homozygotes displayed reduced locomotor activity, and morphine did not induce changes in locomotor activity observed in wild-type mice. Unexpectedly, lack of a functional receptor resulted in changes in both the host defense system and the reproductive system. We observed increased proliferation of granulocyte-macrophage, erythroid, and multipotential progenitor cells in both bone marrow and spleen, indicating a link between hematopoiesis and the opioid system, both of which are stress-responsive systems. Unexpected changes in sexual function in male homozygotes were also observed, as shown by reduced mating activity, a decrease in sperm count and motility, and smaller litter size. Taken together, these results suggest a novel role of the mu opioid receptor in hematopoiesis and reproductive physiology, in addition to its known involvement in pain relief.

Animals↗
PubMed1997

Design, synthesis, and biological activities of four angiotensin II receptor ligands with gamma-turn mimetics replacing amino acid residues 3-5.

Disulfide cyclization is a powerful method for reducing the conformational space of a peptide. This in turn may enable the study of its bioactive conformation. Several analogues of angiotensin II (Ang II) containing a disulfide bridge between amino acids 3 and 5 have been reported. Among these the cyclic octapeptides c[Hcy3,5]-Ang II, c[Cys3,5]-Ang II, and c[Pen 3,5]-Ang II showed significant activity at Ang II receptors. We have performed conformational analysis studies using theoretical calculations and 1H-NMR spectroscopy on tripeptide model compounds of these cyclic octapeptides which show that the cyclic moieties of c[Cys3,5]-Ang II and c[Pen3,5]-Ang II preferentially assume an inverse gamma-turn conformation. On the basis of these results, we substituted amino acid residues 3-5 in Ang II with two different gamma-turn mimetics giving four diastereomeric Ang II analogues. Interestingly, two of these are equipotent to Ang II in binding to AT1 receptors. In the contractile test using rabbit aorta rings, one of the analogues is an agonist with full contractile activity approximately equipotent to c[Pen3,5]-Ang II but 300-fold less potent than Ang II. This low potency may suggest that Ang II does not adopt a gamma-turn in the 3-5 region when interacting with the receptor.

Angiotensin II↗
PubMed1997

Transthyretin quaternary and tertiary structural changes facilitate misassembly into amyloid.

Human transthyretin (TTR) can be transformed into amyloid fibrils by partial acid denaturation to yield a monomeric amyloidogenic intermediate that self-associates into amyloid through quaternary structural intermediates, which are identified by sedimentation velocity methods. The monomeric amyloidogenic intermediate has substantial beta-sheet structure with a nonnative but intact tertiary structure as discerned from spectroscopic methods. Proteolysis sensitivity studies suggest that the C-strand-loop-D-strand portion of TTR becomes disordered and moves away from the core of the beta-sandwich fold upon formation of the monomeric amyloidogenic intermediate over the pH range 5.1-3.9. The single site mutations that are associated with early onset amyloid disease [familial amyloid polyneuropathy (FAP)] function by destabilizing tetrameric TTR. Under mild denaturing conditions, the FAP variants populate the monomeric amyloidogenic intermediate conformation, which assembles into amyloid, whereas wild-type TTR remains tetrameric and nonamyloidogenic. The FAP mutations do not significantly alter the native folded structure; instead, they appear to act by making the thermodynamics and perhaps the kinetics more favorable for formation of the amyloidogenic intermediate. Suppressor mutations have also been characterized that strongly stabilize tetrameric TTR and disfavor the formation of the monomeric amyloidogenic intermediate, thus inhibiting amyloid formation. The mechanistic details characterizing transthyretin amyloid fibril formation available from the biophysical studies outlined within have been utilized to develop a new therapeutic strategy for intervention in human amyloid disease. This approach features small molecules that bind with high affinity to the normal fold of transthyretin, inhibiting the quaternary and tertiary structural changes associated with the formation of the monomeric amyloidogenic intermediate that self-assembles into amyloid. Ligand binding to TTR stabilizes the native tetrameric fold, which is nonamyloidogenic.

Amyloid↗
PubMed1996

Inhibiting transthyretin amyloid fibril formation via protein stabilization.

Transthyretin (TTR) amyloid fibril formation is observed systemically in familial amyloid polyneuropathy and senile systemic amyloidosis and appears to be the causative agent in these diseases. Herein, we demonstrate conclusively that thyroxine (10.8 microM) inhibits TTR fibril formation efficiently in vitro and does so by stabilizing the tetramer against dissociation and the subsequent conformational changes required for amyloid fibril formation. In addition, the nonnative ligand 2,4,6-triiodophenol, which binds to TTR with slightly increased affinity also inhibits TTR fibril formation by this mechanism. Sedimentation velocity experiments were employed to show that TTR undergoes dissociation (linked to a conformational change) to form the monomeric amyloidogenic intermediate, which self-assembles into amyloid in the absence, but not in the presence of thyroxine. These results demonstrate the feasibility of using small molecules to stabilize the native fold of a potentially amyloidogenic human protein, thus preventing the conformational changes, which appear to be the common link in several human amyloid diseases. This strategy and the compounds resulting from further development should prove useful for critically evaluating the amyloid hypothesis--i.e., the putative cause-and-effect relationship between TTR amyloid deposition and the onset of familial amyloid polyneuropathy and senile systemic amyloidosis.

Amyloidosis↗
PubMed1996

The acid-mediated denaturation pathway of transthyretin yields a conformational intermediate that can self-assemble into amyloid.

Transthyretin (TTR) amyloid fibril formation is observed during partial acid denaturation and while refolding acid-denatured TTR, implying that amyloid fibril formation results from the self-assembly of a conformational intermediate. The acid denaturation pathway of TTR has been studied in detail herein employing a variety of biophysical methods to characterize the intermediate(s) capable of amyloid fibril formation. At physiological concentrations, tetrameric TTR remains associated from pH 7 to pH 5 and is incapable of amyloid fibril formation. Tetrameric TTR dissociates to a monomer in a process that is dependent on both pH and protein concentration below pH 5. The extent of amyloid fibril formation correlates with the concentration of the TTR monomer having an altered, but defined, tertiary structure over the pH range of 5.0-3.9. The inherent Trp fluorescence-monitored denaturation curve of TTR exhibits a plateau over the pH range where amyloid fibril formation is observed (albeit at a higher concentration), implying that a steady-state concentration of the amyloidogenic intermediate with an altered tertiary structure is being detected. Interestingly, 1-anilino-8-naphthalenesulfonate fluorescence is at a minimum at the pH associated with maximal amyloid fibril formation (pH 4.4), implying that the amyloidogenic intermediate does not have a high extent of hydrophobic surface area exposed, consistent with a defined tertiary structure. Transthyretin has two Trp residues in its primary structure, Trp-41 and Trp-79, which are conveniently located far apart in the tertiary structure of TTR. Replacement of each Trp with Phe affords two single Trp containing variants which were used to probe local pH-dependent tertiary structural changes proximal to these chromophores. The pH-dependent fluorescence behavior of the Trp-79-Phe mutant strongly suggests that Trp-41 is located near the site of the tertiary structural rearrangement that occurs in the formation of the monomeric amyloidogenic intermediate, likely involving the C-strand-loop-D-strand region. Upon further acidification of TTR (below pH 4.4), the structurally defined monomeric amyloidogenic intermediate begins to adopt alternative conformations that are not amyloidogenic, ultimately forming an A-state conformation below pH 3 which is also not amyloidogenic. In summary, analytical equilibrium ultracentrifugation, SDS-PAGE, far- and near-UV CD, fluorescence, and light scattering studies suggest that the amyloidogenic intermediate is a monomeric predominantly beta-sheet structure having a well-defined tertiary structure.

Amyloid↗
PubMed1996

FAP mutations destabilize transthyretin facilitating conformational changes required for amyloid formation.

Functional transthyretin (TTR) can be transformed into amyloid by partial acid denaturation yielding a monomeric amyloidogenic intermediate which self-associates. The amyloidogenic intermediate has substantial beta-sheet structure with non-native but defined tertiary structure. pH-dependent proteolysis sensitivity studies have identified portions of TTR which become disordered and solvent-exposed in the amyloidogenic intermediate. These include the C-strand-loop D-strand portion of TTR which moves away from the core of the beta-sandwich fold. Mutations that are associated with early onset-amyloid disease (familial amyloidotic polyneuropathy; FAP) function by destabilizing tetrameric TTR in favour of the monomeric amyloidogenic intermediate which has a rearranged C-strand-loop D-strand region. In most cases the FAP mutations do not significantly alter the native folded structure, but instead act on the denaturation pathway by a mechanism that is not completely understood. Interestingly, mutations have also been characterized which strongly stabilize tetrameric TTR and make amyloid formation very difficult at pHs accessible in vivo.

Amyloid↗
PubMed1995

Comparison of lethal and nonlethal transthyretin variants and their relationship to amyloid disease.

The role that transthyretin (TTR) mutations play in the amyloid disease familial amyloid polyneuropathy (FAP) has been probed by comparing the biophysical properties of several TTR variants as a function of pH. We have previously demonstrated that the partial acid denaturation of TTR is sufficient to effect amyloid fibril formation by self-assembly of a denaturation intermediate which appears to be monomeric. Earlier studies on the most pathogenic FAP variant known, Leu-55-Pro, revealed that this variant is much less stable toward acid denaturation than wild-type TTR, apparently explaining why this variant can form amyloid fibrils under mildly acidic conditions where wild-type TTR remains nonamyloidogenic. The hypothesis that FAP mutations destabilize the TTR tetramer in favor of a monomeric amyloidogenic intermediate under lysosomal (acidic) conditions is further supported by the data described here. We compare the acid stability and amyloidogenicity of the most prevalent FAP variant, Val-30-Met, along with the double mutant, Val-30-Met/Thr-119-Met, which serves to model the effects of these mutations in heterozygous patients where the mutations are in different subunits. In addition, we have characterized the Thr-119-Met TTR variant, which is a common nonpathogenic variant in the Portuguese population, to further investigate the role that this mutation plays in protecting individuals who also carry the Val-30-Met mutation against the classically severe FAP pathology. This biophysical study demonstrates that Val-30-Met TTR is significantly less stable toward acid denaturation and more amyloidogenic than wild-type TTR, which in turn is less stable and more amyloidogenic than Thr-119-Met TTR. Interestingly, the double mutant Val-30-Met/Thr-119-Met is very similar to wild-type TTR in terms of its stability toward acid denaturation and its amyloidogenicity. The data suggest that the Thr-119-Met mutation confers decreased amyloidogenicity by stabilizing tetrameric TTR toward acid denaturation. In addition, fluorescence studies monitoring the acid-mediated denaturation pathways of several TTR variants reveal that the majority exhibit a plateau in the relative fluorescence intensity over the amyloid-forming pH range, i.e., ca. pH 4.3-3.3. This intensity plateau suggests that the amyloidogenic intermediate(s) is (are) being observed over this pH range. The Thr-119-Met variant does not exhibit this plateau presumably because the amyloidogenic intermediate(s) do(es) not build up in concentration in this variant. The intermediate is undoubtedly forming in the Thr-119-Met variant, as it will form amyloid fibrils at high concentrations; however, the intermediate is only present at a low steady-state concentration which makes it difficult to detect.

Amino Acid Sequence↗
PubMed1995

Decreased binding of growth hormone in the rat hypothalamus and choroid plexus following morphine treatment.

Male Sprague-Dawley rats were continuously infused with morphine through subcutaneously implanted mini-osmotic pumps over a period of 5 days. The binding of rat growth hormone (rGH) to specific sites in choroid plexus, cortex, hypothalamus, hippocampus and striatum was determined. It was found that in the acute phase of morphine administration the density of growth hormone-binding sites was significantly decreased in choroid plexus and in hypothalamus, but not in any other of the tissues studied. When tolerance to morphine was developed, the level of growth hormone-binding was restored to control level. In the acute phase, the plasma levels of GH, as measured by radioimmunoassay, correlated negatively with the density of the binding sites in choroid plexus and hypothalamus. The decrease in growth hormone-binding in these regions of the rat brain was also confirmed by SDS-polyacrylamide gel electrophoresis of cross-linked complexes of the binding entities to 125I-labelled rGH as visualized by autoradiography. In experiments, where morphine was administrated by intermittent injections, a similar decrease in rGH-binding was observed. However, the time-course of this decrease seemed to be dependent upon the route of administration. Following intracerebroventricular (i.c.v.) injections, the binding of the hormone was already affected after 30 min, whereas the binding of rGH in brain areas after subcutaneous (s.c.) injections was affected at a later stage.

Animals↗
PubMed1995

Morphine-induced prolactin release precedes a down-regulation of prolactin receptors in the male rat choroid plexus and hypothalamus.

In previous studies we provided evidence for changes in prolactin (PRL) receptor levels in the male rat brain after continuously infusing morphine using subcutaneously implanted miniosmotic pumps. In this work we have studied the binding of PRL in the male rat brain following morphine administration by both subcutaneous (s.c.) and intracerebroventricular (i.c.v.) injections. The binding in the choroid plexus and the hypothalamus was measured using iodinated ovine PRL (oPRL) as a radiolabel. The results indicated that the density of the PRL-binding sites in the hypothalamus and the choroid plexus were significantly decreased 4 h and 24 h after s.c. injections, and also 30 min and 4 h after i.c.v. injections. However, no decrease in PRL-binding was observed 15 min after i.c.v. injection of morphine. The plasma levels of PRL were measured by radioimmunoassay (RIA) and were found to be significantly increased after 30 min and 4 h in all treated animals. Following i.c.v. injection a significant increase in plasma PRL was observed after just 15 min. It was suggested that the down-regulation in PRL binding to some extent at least resulted from receptor overstimulation caused by the morphine-induced elevation in the concentrations of the endogenous hormone.

Animals↗
PubMed1994

Effects of morphine on prolactin receptors in the rat brain.

The effect of chronically given morphine on the binding of ovine prolactin (oPRL) to specific areas in the male rat brain was studied. The drug was delivered through subcutaneously implanted miniosmotic pumps. The results indicated that the density of prolactin binding sites in the hypothalamus and the choroid plexus was significantly decreased in the acute phase of morphine administration but restored to control levels when tolerance to morphine was developed. The decrease in prolactin binding was contrasted by elevated plasma levels of the hormone. A negative correlation was found between the hormone concentration in plasma and the density of its binding sites in the hypothalamus and choroid plexus. The hormone-binding sites in these two regions were further characterized with regard to binding constants and molecular sizes. The relevance of the present results with respect to the hypothalamic control of prolactin secretion is discussed.

Animals↗
PubMed1994

Characterization of growth hormone binding sites in rat brain.

The binding of 125I-labelled rat growth hormone (GH) to different areas in the brain was studied in male Sprague-Dawley rats. A high density of GH binding was found in the choroid plexus, hypothalamus, hippocampus, pituitary and spinal cord, whereas a lower binding density was observed in the cortex. Binding of the hormone to the various brain regions was age dependent. Binding was also dependent on time, pH and protein concentration. The binding affinity of the labelled hormone to choroid plexus was 4.3 per nmol/l and the binding capacity was 33.4 nmol/mg protein. The corresponding figures for binding of 125I-labelled GH to hypothalamus were 5.6 per nmol/l and 21.6 nmol/mg protein. By sodium dodecyl sulphate electrophoresis of the cross-linked hormone-receptor complexes, molecular weights of 60,000 and 61,000 were determined for the binding units in the choroid plexus and hypothalamus, respectively. It was further indicated that the binding unit for rat GH was distinct from that for prolactin.

Animals↗
PubMed1994

Proliferation inhibition and CD4/CD8 thymocyte subset skewing by in vivo exposure of C57BL/6 mice to Ah receptor-binding 3,3',4,4'-tetrachlorobiphenyl.

3,3',4,4'-Tetrachlorobiphenyl (TCB) and other Ah receptor-binding xenobiotics lead to thymus atrophy and immunosuppression, the former possibly causing the latter. In order to better understand the TCB-induced events in the murine thymus, we analyzed the effects of TCB on the proliferation capacity and maturation kinetics of different thymocyte subsets in 2-week-old C57BL/6 mice (i.e. of the Ahb-1 'dioxin-sensitive' genotype). Mice were injected with a single dose of TCB, and the development of thymocytes was followed up for 10 days using flow cytometric surface marker analysis combined with measurement of DNA content by 7-amino-actinomycin D staining. Already 2 days after exposure to TCB, fewer of the more immature thymocytes (CD4-CD8-, CD4-CD8+ alpha beta TCR-) were proliferating than in thymi from control animals. Eventually this led to a severe decrease in thymus cellularity. Moreover, a shift towards the CD4-CD8+ mature subpopulation was observed. The effects were reversible, and proliferation and CD4/CD8 subset distribution returned to normal levels within the observation period. The results are in good agreement with the data obtained previously in vitro with fetal thymus organ cultures.

Animals↗
PubMed1993

Age-related reduction of human growth hormone-binding sites in the human brain.

Previous studies have shown that alterations in various neuroendocrine functions occur with increasing age. We here report a study of growth hormone (GH)-binding sites in different areas of post-mortem human brains collected from individual males and females of different age. The results indicate that there exists a significant negative correlation between the density of GH-binding sites and increasing age. This phenomenon was observed in both sexes in brain areas such as choroid plexus, hippocampus, hypothalamus, pituitary and putamen but not in e.g. thalamus. In all tissues (except for choroid plexus), the GH binding was significantly higher in those originating from females than those from males. This discrepancy was found likely to be associated with the affinity of GH to lactogenic rather than to somatogenic sites as no pronounced sex difference in binding was observed in the presence of excessive amounts of human prolactin. Data also indicate that the putative GH receptors in the various brain regions differ with regard to binding constants and to the estimated molecular size of the hormone-binding units. The loss of GH receptors in brain of elderly people may have consequences in several physiological courses. The decrease in GH binding at hypothalamic and pituitary levels may be of importance for the mechanisms behind the release or secretion of the hormone.

Adult↗
PubMed1992

Characterization of prolactin receptors in human choroid plexus.

The specific binding of 125I-human prolactin (hPRL) was studied in different areas of the human brain. Particularly high binding affinity of the hormone was found in the choroid plexus and this tissue was therefore selected for further studies. The hippocampus, the hypothalamus and the pituitary were among other regions containing prolactin-binding sites. In the choroid plexus the amount of PRL receptors was significantly higher in females than in males and was also found in both sexes to decrease with age. The binding affinity of 125I-hPRL to choroid plexus was 3.0 x 10(9) M-1 and the binding capacity was 10.3 pmol per mg protein. Following solubilization with Triton X-100 the PRL receptor fraction retained its hormone-binding properties and upon molecular sieve chromatography it behaved as a protein with a molecular mass of approximately 250,000. Cross-linking of 125I-hPRL to receptors from choroid plexus and subsequent sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis indicated a major hormone-binding unit of M(r) 44,000. This value is about 7,000 smaller than that reported earlier by us for the growth hormone receptors from the same tissue, following cross-linking to 125I-human growth hormone (hGH). By affinity column chromatography a complete separation of the hPRL and hGH binding units was achieved. It was thus shown that in choroid plexus the binding sites for GH and PRL occur as discrete entities.

Aging↗
PubMed1991

The role of oxygen free radicals in myocardial ischemia/reperfusion injury.

A series of experiments have been done to investigate the role of oxygen free radicals in ischemia/reperfusion injury. The following results were found: Myocardial MDA content increased significantly after post-ischemic reperfusion in vivo and in vitro. A blockade of the xanthine oxidase pathway for free radical generation could provide effective protection against ischemia/reperfusion injury. Exogenous reactive oxygen intermediates H2O2, .OH and O2- could induce changes in the contractility and electrophysiological properties of myocardial cells similar to those seen in ischemia/reperfusion. An outburst of free radical generation was detected by ESR spectroscopy at low temperature (-173 degrees C) and with the spin trapping technique during the very early phase of reperfusion. The authors emphasize the important role of free radicals in the pathogenesis of myocardial ischemia/reperfusion injury.

Action Potentials↗
PubMed1991

Role of amygdaloid nucleus in the correlation between the heart and the acupoint neiguan in rabbits.

Amygdaloid nucleus (AMYG) could converge and integrate the signals from electroacupuncture (EA) at Neiguan (P 6) and acute myocardial ischemia (AMI) in rabbits. And EA could regulate the changes in the discharge of single units in AMYG caused by AMI, that is, EA inhibited the increased frequency, and reversed the decreased frequency. This suggested that AMYG is an important link in correlation between the heart and the acupoint Neiguan, providing an experimental basis for the Theory of Channel--Viscera Correlation.

Acupuncture Points↗