Design, activity, and 2.8 A crystal structure of a C2 symmetric inhibitor complexed to HIV-1 protease.

A two-fold (C2) symmetric inhibitor of the protease of human immunodeficiency virus type-1 (HIV-1) has been designed on the basis of the three-dimensional symmetry of the enzyme active site. The symmetric molecule inhibited both protease activity and acute HIV-1 infection in vitro, was at least 10,000-fold more potent against HIV-1 protease than against related enzymes, and appeared to be stable to degradative enzymes. The 2.8 angstrom crystal structure of the inhibitor-enzyme complex demonstrated that the inhibitor binds to the enzyme in a highly symmetric fashion.

Social learning and teenage drug use: an analysis of family dyads.

This analysis of drug use in family dyads draws upon data from a series of nationwide studies in which interviews were conducted separately with teenagers and with older members of their families, i.e., their mothers, fathers, and older siblings. The interview schedule for these studies examines each individual's personal experience with a broad range of psychoactive substances. Thus, to the extent that behavioral similarities do occur in family dyads, "same drug links" can be compared to "cross drug links," providing a basis for differentiating evidence of direct imitation from less specific patterns of behavioral similarity. The theoretical implications of these findings are discussed.

Using eye movement desensitization and reprocessing to treat complex PTSD in a biracial client.

A biracial client's recovery from posttraumatic stress disorder (PTSD) through the use of eye movement desensitization and reprocessing (EMDR) is discussed to illustrate the interaction between ethnicity and phenotype as well as diagnosis and treatment considerations. This case explores a woman's experience of discrimination in and out of her home and her vulnerability to complex PTSD, and it documents the importance of the therapy focusing on experiences of discrimination and prejudice as well as abuse. It shows how the client structures her environment in a personally creative fashion to include representative features of various aspects of her identity, by her choice of where and who she teaches as well as how and with whom she spends her free time.

A cojoint, operant model for assisting profoundly behaviorally disordered adolescents.

This article describes a community treatment program established in a semirural New Mexico community that was cojointly administered by the local school district, university and mental health center in order to facilitate the adoption of personal and academic skills among profoundly, behaviorally disordered adolescents. The treatment, a model classroom, is staffed by a fulltime teacher and a full-time psychologist whose roles are differentiated primarily by the ways and type of operant behavior they reinforce. This cojoint operant model for assisting profoundly behaviorally disordered adolescents was successful in facilitating the overall adjustment of eight adolescents in one academic year, as evidenced results of a behavior rating scale, projective testing, and parents' checklist and individual interviews. Theoretical bases and procedures for implementing the model are described, and recommendations for future programs serving emotionally disturbed adolescents are proposed.

Peptide mapping by polyacrylamide gel electrophoresis after cleavage at aspartyl-prolyl peptide bonds in sodium dodecyl sulfate-containing buffers.

Protein samples prepared for sodium dodecyl sulfate-polyacrylamide gel electrophoresis are preferentially cleaved at aspartyl-prolyl peptide bonds upon heating at 110 degrees C. The presence of aspartyl-prolyl peptide bonds in a protein can therefore be detected by gel electrophoresis of heated samples and the resulting peptides mapped. The method of heat cleavage also works well with proteins in bands cut from electrophoresed gels using modified stacking conditions in the second electrophoresis. An immunoblotting procedure for peptide mapping of nanogram quantities of specific proteins in complex mixtures is demonstrated. Peptide maps produced by aspartyl-prolyl peptide bond cleavage of fructose-1,6-bisphosphatases from different sources show the effectiveness of the above techniques and suggest a conservation of aspartyl-prolyl peptide bonds in pig kidney and mouse and rat liver fructose-1,6-bisphosphatases.

Personality and physical health: an introduction.

This article provides a rationale for research on personality as a contributing factor in the development of physical disease. A brief history of major developments from the 1930s to the present is then provided. Special attention is given to shifts in conception regarding whether particular dispositions are related to specific physical disorders or whether these dispositions increase general illness susceptibility. The paper ends with a brief orientation to the other papers and commentaries in this special issue.

Phosphorylation in vivo of yeast (Saccharomyces cerevisiae) fructose-1,6-bisphosphatase at the cyclic AMP-dependent site.

In vivo labeled fructose-1,6-bisphosphatase was immunopurified from yeast (Saccharomyces cerevisiae) cells that had been incubated in the presence of [32P] orthophosphate. Tryptic peptides from labeled enzyme were mapped by high performance liquid chromatography. Most of the radioactivity was found to be associated with the peptide Arg9 through Arg24, the same peptide which had been previously shown to be phosphorylated in vitro by cAMP-dependent protein kinase (Rittenhouse, J., Harrsch, P. B., Kim, J. N., and Marcus, F. (1986) J. Biol. Chem. 261, 3939-3943). The amino acid sequence analysis suggests that phosphorylation occurs at the same site, Ser11. We have also determined the extent of phosphorylation at Ser11 of fructose-1,6-bisphosphatase in yeast cultures growing under various nutritional conditions by measuring the relative amounts of phospho- and corresponding dephosphopeptides in tryptic digests. Significant levels of phosphorylation of the enzyme were found in yeast cultures grown under gluconeogenic conditions that varied from 0.15 to 0.50 mol of phosphate per mol of enzyme subunit. However, phosphate incorporation rapidly increased to greater than 0.8 mol after addition of glucose to these cultures. An alternative technique, based solely on enzyme activity measurements, was also developed to estimate the extent of fructose-1,6-bisphosphatase phosphorylation in yeast cultures. The results obtained with this technique agreed with those obtained by high performance liquid chromatography of tryptic peptides.

Distribution of a COOH-terminal amino acid extension of liver fructose-1,6-bisphosphatase among rodent species.

We have recently established from sequence analysis that rat liver fructose-1,6-bisphosphatase contains a 24-26 residue extension beyond the COOH-terminal amino acid of other mammalian fructose-1,6-bisphosphatases that results in an increased subunit molecular weight (Rittenhouse et al. (1983) J. Biol. Chem. 258, 7648-7652). In the present work the distribution of the COOH-terminal extension of fructose-1,6-bisphosphatases was tested by subunit molecular weight analysis of the enzyme immunoprecipitated from liver extracts. Of all rodent species tested, including several Muridae other than Rattus; only the enzyme from animals of the genus Rattus was found to have the extension. Further studies on the distribution of the enzyme extension could provide a simple tool to study the phylogeny of the genus Rattus.

Inhibition of Escherichia coli fructose-1,6-bisphosphatase by fructose 2,6-bisphosphate.

Fructose 2,6-bisphosphate, a potent inhibitor of fructose-1,6-bisphosphatases, was found to be an inhibitor of the Escherichia coli enzyme. The substrate saturation curves in the presence of inhibitor were sigmoidal and the inhibition was much stronger at low than at high substrate concentrations. At a substrate concentration of 20 microM, 50% inhibition was observed at 4.8 microM fructose 2,6-bisphosphate. Escherichia coli fructose-1,6-bisphosphatase was inhibited by AMP (Ki = 16 microM) and phosphoenolpyruvate caused release of AMP inhibition. However, neither AMP inhibition nor its release by phosphoenolpyruvate was affected by the presence of fructose 2,6-bisphosphate. The results obtained, together with previous observations, provide further evidence for the fructose 2,6-bisphosphate - fructose-1,6-bisphosphatase active site interaction.

Phosphorylation in situ of atrial natriuretic peptide prohormone at the cyclic AMP-dependent site.

Previously we have shown that atrial natriuretic peptides (ANP) are phosphorylated at Ser-104 by cyclic AMP-dependent protein kinase (Rittenhouse, J. Moberly, L., O'Donnell, M.E., Owen, N.E., and Marcus, F. (1986) J. Biol. Chem. 261, 7607-7610). In our present study, atrial natriuretic peptide prohormone (pro-ANP) purified from extracts of rat atria that had been incubated in situ with [32P]orthophosphate was found to be phosphorylated. The site of in situ phosphorylation was localized to the ANP region of the prohormone and was further delineated to a chymotryptic peptide having the same retention time by high performance liquid chromatography as the hexapeptide Arg101-Arg102-Ser103-Ser(P)104-Cys105-Phe106+ ++. This hexapeptide was also formed from in vitro phosphorylated pro-ANP and synthetic ANP. It is thus likely that Ser-104 is the site of phosphorylation of pro-ANP both in situ and in vitro. Incubation of atria with intracellular cyclic AMP-elevating agents consistently increased the specific radioactivity of the resulting purified pro-ANP by 5-10-fold. However, the overall extent of phosphorylation was low, suggesting that a subpopulation of pro-ANP molecules are phosphorylated at Ser-104 by a cyclic AMP-mediated pathway.

Chinese medicinal herbs reverse macrophage suppression induced by urological tumors.

Tumors are known to produce factors suppressing macrophage function. In this study we demonstrated in vitro suppression of macrophage chemiluminescent oxidative burst associated with viable cells and cell-free extracts of two urological neoplasms--murine renal cell carcinoma (Renca) and murine bladder tumor (MBT). Suppression was reversed by extracts of two Chinese medicinal herbs, Astragalus membranaceus (AM) and Ligustrum lucidum (LL). Murine macrophage cell line J774 was incubated with either the viable tumor cells or the cell-free tumor extract for 18 hours at 37C and 5% CO2. Chemiluminescent oxidative burst as an indicator of macrophage function was triggered by adding zymosan A suspension containing luminol and assayed in an automated luminometer. Photon emission over time was counted and the results were expressed as integrated photon emission. Significant dose-related depression of oxidative burst occurred with either the viable tumor cells or the cell-free tumor extracts. Depression was partially or completely reversed by the presence of 50-100 micrograms./ml. of either the AM or the LL extract. AM and LL have previously been shown to modulate immune response. Data from this study suggest that they may also exert their antitumor activity via abolition of tumor-associated macrophage suppression.

A re-evaluation of the molecular weight of yeast (Saccharomyces cerevisiae) fructose-1,6-bisphosphatase.

In contrast with previous results that indicate that Saccharomyces cerevisiae fructose-1,6-bisphosphatase is a dimer of 56,000 molecular weight subunits, we find that the subunit Mr of the enzyme purified from baker's yeast is 40,000. The same subunit Mr was observed in immunoprecipitates of crude supernatants of baker's yeast and S. cerevisiae cultures, as well as in acid-extracts of cells detected by immunoblotting, suggesting that the native subunit indeed has a Mr of 40,000 and it has not been produced from a larger polypeptide. Complete immunoprecipitation of fructose-1,6-bisphosphatase activity with saturating concentrations of specific antibody suggests that there is only one fructose-1,6-bisphosphatase isozyme in S. cerevisiae. The Mr of the purified enzyme determined by size exclusion HPLC suggests that it has a tetrameric structure characteristic of fructose-1,6-bisphosphatases from a broad phylogenetic spectrum.

Amino acid sequence homology among fructose-1,6-bisphosphatases.

The hydrolysis of fructose 1,6-bisphosphate to fructose 6-phosphate is a key reaction of carbohydrate metabolism. The enzyme that catalyzes this reaction, fructose-1,6-bisphosphatase, appears to be present in all forms of living organisms. Regulation of the enzyme activity, however, occurs by a variety of distinct mechanisms. These include AMP inhibition (most sources), cyclic AMP-dependent phosphorylation (yeast), and light-dependent activation (chloroplast). In the present studies, we have made a comparison of the primary structure of mammalian fructose-1,6-bisphosphatase with the sequence of peptides isolated from the yeast Saccharomyces cerevisiae, Escherichia coli, and spinach chloroplast enzymes. Our results demonstrate a high degree of sequence homology, suggesting a common evolutionary origin for all fructose-1,6-bisphosphatases.

Function, structure and evolution of fructose-1,6-bisphosphatase.

The hydrolysis of fructose-1,6-bisphosphate to fructose-6-phosphate is a key reaction of carbohydrate metabolism. The enzyme that catalyzes this reaction, fructose-1,6-bisphosphatase, appears to be present in all forms of living organisms. Regulation of the enzyme activity, however, occurs by a variety of distinct mechanisms. These include AMP inhibition (most sources), cyclic AMP-dependent phosphorylation (yeast), and light-dependent activation (chloroplast). In this short review, we have analyzed the function of several fructose-1,6-bisphosphatases and we have made a comparison of partial amino acid sequences obtained from the enzymes of the yeast Saccharomyces cerevisiae, Escherichia coli, and spinach chloroplasts with the known entire amino acid sequence of a mammalian gluconeogenic fructose-1,6-bisphosphatase. These results demonstrate a very high degree of sequence conservation, suggesting a common evolutionary origin for all fructose-1,6-bisphosphatases.

Yeast (Saccharomyces cerevisiae) fructose-1,6-bisphosphatase. Properties of phospho and dephospho forms and of two mutants in which serine 11 has been changed by site-directed mutagenesis.

The properties of dephospho- and phosphofructose-1,6-bisphosphatase from the yeast Saccharomyces cerevisiae and of two mutant enzymes in which the phosphorylatable Ser11 had been changed by site-directed mutagenesis (Ser----Ala and Ser----Asp) were studied to clarify the role of cyclic AMP-dependent phosphorylation of yeast fructose-1,6-bisphosphatase. The mutant enzymes and wild type Ser11 fructose-1,6-bisphosphatase were overexpressed and purified to homogeneity. Phosphofructose-1,6-bisphosphatase was prepared by in vitro phosphorylation. The comparison of the properties of the above enzymes demonstrated that all four had similar maximum activity. However, the phosphoenzyme was about 3-fold more sensitive to AMP and fructose 2,6-bisphosphate inhibition than the dephosphoenzyme, suggesting that regulation operates in vivo by this mechanism, leading to decreased enzyme activity. The purified mutant enzymes Ala11 and Asp11 exhibited properties closely similar to those of dephospho- and phosphofructose-1,6-bisphosphatase, respectively. These results indicate that the functional group at residue 11 is an important factor in the regulation of fructose-1,6-bisphosphatase activity and that Ser(P) can be functionally substituted by Asp in this enzyme.

Regulation of astrocytic tenascin by basic fibroblast growth factor.

Extracellular matrix (ECM) molecules have been implicated in the regulation of neuronal adhesion and neurite outgrowth both during development and after injury. It has been demonstrated in our laboratory that astrocytes are heterogeneous in expression of the ECM molecule tenascin. High-tenascin astrocytes have a reduced ability to support neurite outgrowth. In addition, astrocytes treated with exogenous basic fibroblast growth factor (bFGF) supported reduced neuronal growth and adhesion. In the current study, the hypothesis was tested that bFGF could increase the expression of tenascin by these cells. Basic FGF was added to cultures of rat cerebral cortical astrocytes at concentrations of up to 30 ng/ml, concentrations shown to have a significant effect on neuronal adhesion. Tenascin levels were evaluated by Western blot analysis of both cell extracts and conditioned media and also by immunocytochemistry techniques. Tenascin levels began to increase after 24-48 hr and continued to increase throughout 8 days in culture. The increase in tenascin was concentration-dependent, with the largest increase seen at 5 ng/ml bFGF. Tenascin production was increased approximately 5.5-fold in serum-containing medium but only about 2-fold in serum-free medium. When heparin (10 micrograms/ml) was included along with bFGF in serum-free medium, tenascin production was further enhanced. The bFGF treatment was discontinued after 8 days, and the cells were maintained for an additional 8 days in culture. Tenascin levels returned to control values, demonstrating that the bFGF effect is transient. It is our hypothesis that the action of bFGF during injury may evoke the induction of tenascin on astrocytes, thereby reducing regeneration in the central nervous system.

Serum ornithine carbamyl transferase activities in Reye's syndrome.

Serum levels of ornithine carbamyl transferase (S-OCT), glutamic oxaloacetic transaminase and glutamic pyruvic transaminase were compared for 37 Reye's syndrome patients with regard to variation with clinical stage an serum ammonia levels. In stage I patients, the mean S-OCT activities were greater and the serum ammonia levels lower than found with patients in the more advanced stages. Covariation of these two parameters was found only in the more advanced stages. No significant correlation with stage or serum ammonia levels was found for S-GOT or S-GPT activities. These observations are discussed in terms of their relevance to reports of an early transient decrease of hepatic OCT activity in Reye's syndrome.

Development of sensitive immunoassays to detect amylin and amylin-like peptides in unextracted plasma.

Amylin is a 37-amino-acid polypeptide synthesized in and secreted from pancreatic beta cells along with insulin. Its biological actions include the slowing and reduction of postmeal increases in plasma glucose concentrations. Studies of the basic amylin biology in humans have been hampered by the lack of a rapid, sensitive assay capable of measuring physiological concentrations of amylin in small volumes of plasma. We report here two sandwich-type immunoassays that use pairs of monoclonal antibodies, the fluorescent substrate 4-methylumbelliferyl phosphate, and the enzyme alkaline phosphatase. The minimum detectable concentration of amylin in 50 microL of plasma was 0.5 to 2 pmol/L, and the dynamic range was 2 to 100 pmol/L. The assays had average intraassay CVs of <10%, average interassay CVs of <15%, and good linearity on dilution and recovery of added amylin. The two assays use the same detection antibody, which binds to the carboxyl terminus of the molecule, but different capture antibodies. One of the assays measures only human amylin; the other also detects amylin-like peptides. Examples of measurements in human plasma are provided in subjects with impaired glucose tolerance and in nondiabetic controls.

Amino acid sequence of the phosphorylation site of yeast (Saccharomyces cerevisiae) fructose-1,6-bisphosphatase.

Fructose-1,6-bisphosphatase from the yeast Saccharomyces cerevisiae has properties similar to other gluconeogenic fructose-1,6-bisphosphatases, but an unusual characteristic of the yeast enzyme is that it can be phosphorylated in vitro by cAMP-dependent protein kinase. Phosphorylation also occurs in vivo, presumably as part of a signalling mechanism for the enzyme's degradation. To probe the structural basis for the phosphorylation of yeast fructose-1,6-bisphosphatase, we have developed an improved procedure for the purification of the enzyme and then performed sequence studies with the in vitro-phosphorylated protein as well as with tryptic and chymotryptic peptides containing the phosphorylation site. As a result of these studies, we have determined that yeast fructose-1,6-bisphosphatase has the following 24-residue NH2-terminal amino acid sequence: Pro-Thr-Leu-Val-Asn-Gly-Pro-Arg-Arg-Asp-Ser-Thr-Glu-Gly- Phe-Asp-Thr-Asp-Ile-Ile-Thr-Leu-Pro-Arg. The site of phosphorylation is located at Ser-11 in the above sequence. The amino acid sequence around the site of phosphorylation contains the sequence - Arg-Arg-X-Ser- associated with many of the better substrates of cAMP-dependent protein kinase. The sequence of residues 15-24 above is highly homologous with the sequence of residues 6-15 of pig kidney fructose-1,6-bisphosphatase, showing 7 out of 10 residues in identical positions. The yeast enzyme, however, has a dissimilar NH2-terminal region which extends beyond the NH2 terminus of mammalian fructose-1,6-bisphosphatases and contains a unique phosphorylation site.