Affinity-labeling steroids as biologically active probes of antiglucocorticoid hormone action

The role of the glucocorticoid receptor in the expression of antiglucocorticoid action has been investigated with a chemically-reactive derivative of three glucocorticoid steroids with differing biological potencies, i.e. the C-21 mesylates of cortisol, dexamethasone and deacylcortivazol. Dexamethasone 21-mesylate (Dex-Mes) was the most useful derivative due to its favorable balance of high receptor affinity and predominantly irreversible antiglucocorticoid activity. A number of criteria have been used to conclude that [3H]Dex-Mes covalently labels glucocorticoid receptors in the steroid-binding cavity. The available data indicate that covalent Dex-Mes-labeled receptors (mol. wt approximately equal to 98,000) are responsible for the irreversible antiglucocorticoid activity while the partial agonist activity of Dex-Mes is due to non-covalent Dex-Mes-bound receptors. Further support for this hypothesis comes from the observations that deacylcortivazol 21-mesylate was a full glucocorticoid and did not affinity label receptors (and marginally labeled cytosol proteins) although it was capable of covalently-labeling bovine serum albumin. Several mechanisms for the expression of irreversible antiglucocorticoid activity by covalent Dex-Mes-labeled receptors were examined and can be eliminated. Covalent receptor-Dex-Mes complexes formed in whole HTC cells were found to have a decreased capacity for nuclear binding. This decreased nuclear-binding capacity could be responsible for the whole-cell irreversible antiglucocorticoid activity of Dex-Mes.     

The use of enzymes for searching for biologically active compounds

Possible use of extracellular staphylococcal DNAase in screening substances with potential antibacterial activity was studied on quinoxalin as an example. For screening substances with antiviral activity the possible use of the influenza virus neuraminidase was studied on fluoren as an example. Close correlation between the biological activity of quinoxalin derivatives and the ability to inhibit DNAase was revealed. The most active inhibitors of the enzyme were dioxidin and other biologically active analogs of quinoxalin 1,4-di-N-oxide. The use of the extracellular nuclease as a biochemical model permitted to establish the structure/function dependence with respect to the quinoxalin derivatives. The effect of the fluoren derivatives on activity of the influenza virus neuramididase was studied. It was shown that florenal, an antiviral drug inhibited the virus specific enzyme by 80 to 90 per cent and had no effect on catalytic activity of bacterial neuraminidase. Biologically inactive and slightly active derivatives of the compounds did not inhibit the influenza virus enzyme. At the same time some of them lowered the activity of the bacterial enzyme.     

Receptor-based assays in screening for biologically active substances.

Molecular biology has identified new receptors and ligands which are deregulated in diseases such as cancer and autoimmune conditions and which provide rational targets for therapeutic intervention. Advances in instrumentation and methodology make it possible to screen large numbers of samples in simple receptor-ligand binding assays in the search for drug candidates. Caution must be exercised in the interpretation of data derived from such assays. This is particularly pertinent to the recently characterized receptors, such as the cytokine receptors, as we do not fully understand the relationship between the receptor type and the linkage of receptors to the appropriate or inappropriate second messenger systems that are used in the experimental screening protocols and the disease state.     

Lipotropin: precursor to two biologically active peptides.

Lipotropin appears to be the common precursor to β-MSH, a peptide with lipolytic activity, and C-Fragment, a peptide with potent opiate activity. The product formed is determined by the specificity of the activating enzymes.The amino acid sequence of β-MSH, the 18 residue melanocyte stimulating hormone, is contained within the central region of lipotropin (LPH), a 91 residue polypeptide. On this basis Li and his colleagues¹ suggested that LPH might be the prohormone of β-MSH. Bertagna, Lis and Gilardeau², on the other hand, were unable to demonstrate conversion of LPH to β-MSH $\text{in vitro}$ using pulse labelling techniques. If LPH is the precursor of β-MSH, formation of the hormone should be accompanied by release of the contiguous fragments of the prohormone and the fragments remain in the secretory particle of the gland. To obtain evidence on the biosynthetic origin of β-MSH, we have isolated peptides from pituitary in a search for the N- and C-fragments of the prohormone.     

Exogenously administered HGF activator augments liver regeneration through the production of biologically active HGF.

Hepatocyte growth factor (HGF) plays a crucial role in the recovery of injured liver. Liver functions are mostly impaired in patients with liver diseases including cirrhosis. However, a significant amount of inactive HGF precursor (proHGF) is reported in the plasma of these patients. proHGF is proteolytically converted to an active form (mature HGF) by HGF-activator. Thus conversion of proHGF into mature HGF presumably contributes to the recovery of liver functions. In this study, rats with a partial hepatectomy were used, as proHGF is accumulated in the remnant liver. Recombinant human HGF-activator was administered via the portal vein to investigate the effect on molecular forms of HGF and its biological signaling. rhHGF-activator promptly converted proHGF into mature HGF, reaching maximal levels at 5-10 min after the injection, while the decreased proHGF was quickly recovered to the initial levels in the liver. The HGF receptor/c-Met was found to be autophosphorylated in the liver treated with rhHGF-activator. Further, the proliferating cell nuclear antigen labeling index and the liver regeneration rate were significantly higher in rhHGF-activator group than in control animals. These results indicate that exogenously administered HGF-activator produces a biologically active HGF from its precursor form and increases the potential for liver regeneration in vivo.     

Potential use of exogenous RNAs for the analysis of the complex effects of biologically active substances

It has been established in rat experiments that RNA obtained from the liver and renal cortex of animals given hydrocortisone produces in recipients the rise of physical endurance evoked by the hormone. RNA obtained from other organs of these animals and from any test organs of control donors did not influence physical endurance. The key role of the liver and kidneys in the realization of the hydrocortisone effect is likely to be connected with activation of the synthesis of gluconeogenesis enzymes. RNA obtained from the organs of donors premedicated with hydrocortisone reproduced stimulation of gluconeogenesis with hydrocortisone. The use of exogenous RNA holds promise for analysis of complex effects of biologically active substances, since it permits studying separately the components of the effects determined by activation of protein synthesis in definite organs.     

Preparation of biologically active radioiodinated cholecystokinin for radioreceptor assay and radioimmunoassay.

A modified method is described for the preparation of stable, high specific activity radioiodinated cholecystokinin (CCK) by its conjugation to 125I-Bolton Hunter reagent (125I-BH). Purified radioiodinated CCK (125I-BH-CCK) stimulated amylase release from isolate rat pancreatic acini with a potency identical to that of native CCK. Further, 125I-BH-CCK was highly immunoreactive and reacted with antisera directed toward both the NH2- and COOH-terminal portions of the CCK molecule. Finally, 125I-BH-CCK bound to specific receptor sites on isolated pancreatic acini.     

The algorithm for cross-evaluation (ACE) of biologically active compounds.

A non-linear algorithm, ACE (Algorithm for Cross-Evaluation), was developed to correlate the activity of anti-cancer drugs against human cell lines. ACE evaluates the relationships among compounds and cell lines and displays the resulting clusters on a 3-D surface plot. This plot along with a frequency plot and rank graph provides clear information regarding the sensitivity of groups of cell lines to particular compounds. The program was tested on published anti-cancer data sets and found associations useful in the selection and design of chemotherapy drugs for pre-clinical trials. ACE may be applied to other situations where details of groupings and relationships need to be extracted from large sets of data.     

A biologically active acid hydrolysis product of saxitoxin.

The preparation, purification, and biological and chemical properties of a decarbamylated product of saxitoxin are described.     

The biologically active isomers of conjugated linoleic acid.

Numerous physiological effects are attributed to conjugated linoleic acid (CLA). The purpose of this presentation is to consider these effects with respect to the cis-9,trans-11 and trans-10,cis-12 CLA isomers. We review previously published data and present new findings that relate to underlying biochemical mechanisms of action. Both isomers are natural products. The cis-9,trans-11 isomer is the principal dietary form of CLA, but the concentrations of this isomer and the trans-10,cis-12 isomer in dairy products or beef vary depending on the diet fed to cows or steers, respectively. The trans-10,cis-12 CLA isomer exerts specific effects on adipocytes, in particular reducing the uptake of lipid by inhibiting the activities of lipoprotein lipase and stearoyl-CoA desaturase. The trans-10,cis-12 CLA isomer also affects lipid metabolism in cultured Hep-G2 human liver cells, whereas both the cis-9,trans-11 and trans-10,cis-12 CLA isomers appear to be active in inhibiting carcinogenesis in animal models. We present new findings indicating that the cis-9,trans-11 CLA isomer enhances growth and probably feed efficiency in young rodents. Accordingly, the effects of CLA on body composition (induced by trans-10,cis-12 CLA) and growth/feed efficiency (induced by cis-9,trans-11 CLA) appear to be due to separate biochemical mechanisms. We also show that a 19-carbon CLA cognate (conjugated nonadecadienoic acid, CNA) inhibits lipoprotein lipase activity as effectively as CLA in cultured 3T3-L1 adipocytes. Presumably, CNA is metabolized differently than the 18-carbon CLA isomers, so this finding indicates direct activity of the administered compound as opposed to acting via a metabolite.     

The use of gel chromatography for the isolation of biologically active toxin complex from Bacillus anthrax

The multistep fractioning of the protein components from cultural filtrate of B. anthracis grown on casaminoacids containing medium was done. The scheme for preliminary purification of a toxin complex of B. anthracis against low and high molecular mass contaminants has been elaborated and includes ultrafiltration, gel chromatography in ultragel AcA-202 and TSK-gel toyopal HW-55. Biological activities of the complex,toxicity for laboratory animals and adenylate cyclase activity characteristic of B. anthracis toxin, are shown to remain intact in course of preliminary purification. Molecular masses of protein subunits from the fraction containing anthracis toxin activity reach 85-90 kD, 0.3-0.5 mg of toxin complex protein is yielded from 1 l of B. anthracis cultural filtrate.     

Production of biologically active thymulin in Escherichia coli through expression of a chemically synthesized gene

Summary A synthetic gene coding for thymulin was ligated into an expression vector (pJB 1301) and placed under lac operon control. In the recombinant clones, thymulin was expressed as part of a galactosidase chimeric protein which was then cleaved by cyanogen bromide. Thymulin was purified using various chromatography systems including gel filtration and HPLC, and was detected by radioimmunoassay (RIA). In the biological assay the purified recombinant peptide demonstrated the same zinc dependency as natural thymulin and had the same amino acid composition and primary structure.     

Pleiotypic mechanisms of cellular responses to biologically active lysophospholipids

The activities of cell-derived lysophospholipid (LPL) growth factors on cellular proliferation and a range of proliferation-independent functions are regulated at multiple levels. This section focuses first on the capacity of the actin-severing protein gelsolin to bind lysophosphatidic acid (LPA), but not sphingosine 1-phosphate (S1P), and either sequester LPA or present it to responsive cells. Expression of members of the family of endothelial differentiation gene-encoded G protein-coupled receptors (Edg Rs) for LPLs is controlled developmentally and by cell-activating stimuli. Edg R transduction of cellular effects of LPLs involves both direct actions on target cells and induction of generation of proteins with relevant actions capable of amplifying or diminishing primary direct effects of LPLs. These general mechanisms are evident in Edg R mediation of proliferation, cytokine secretion and suppression of apoptosis. The availability of functionally-active anti-Edg R antibodies and Edg R-specific pharmacological probes, establishment of Edg R transgenes and gene knockouts, and identification of natural genetic anomalies of LPL metabolism and recognition by Edg Rs will permit elucidation of the in vivo activities of LPA and S1P normally and in disease states.     

Pleiotypic mechanisms of cellular responses to biologically active lysophospholipids

The activities of cell-derived lysophospholipid (LPL) growth factors on cellular proliferation and a range of proliferation-independent functions are regulated at multiple levels. This section focuses first on the capacity of the actin-severing protein gelsolin to bind lysophosphatidic acid (LPA), but not sphingosine 1-phosphate (S1P), and either sequester LPA or present it to responsive cells. Expression of members of the family of endothelial differentiation gene-encoded G protein-coupled receptors (Edg Rs) for LPLs is controlled developmentally and by cell-activating stimuli. Edg R transduction of cellular effects of LPLs involves both direct actions on target cells and induction of generation of proteins with relevant actions capable of amplifying or diminishing primary direct effects of LPLs. These general mechanisms are evident in Edg R mediation of proliferation, cytokine secretion and suppression of apoptosis. The availability of functionally-active anti-Edg R antibodies and Edg R-specific pharmacological probes, establishment of Edg R transgenes and gene knockouts, and identification of natural genetic anomalies of LPL metabolism and recognition by Edg Rs will permit elucidation of the in vivo activities of LPA and S1P normally and in disease states.