Development of potent glucagon antagonists: structure-activity relationship study of glycine at position 4.

We examined the functional role of glycine at position 4 in the potent glucagon antagonist [desHis(1), Glu(9)]glucagon amide, by substituting the L- and D-enantiomers of alanine and leucine for Gly(4) in this antagonist. The methyl and isobutyl side-chain substituents were introduced to evaluate the preference shown by the glucagon receptor, if any, for the orientation of the N-terminal residues. The L-amino acids demonstrated only slightly better receptor recognition than the D-enantiomers. These results suggest that the Gly(4) residue in glucagon antagonists may be exposed to the outside of the receptor. The enhanced binding affinities of analogs 1 and 3 compared with the parent antagonist, [desHis(1), Glu(9)]glucagon amide, may have resulted from the strengthened hydrophobic patch in the N-terminal region and/or the increased propensity for a helical conformation due to the replacement of alanine and leucine for glycine. Thus, as a result of the increased receptor binding affinities, antagonist activities of analogs 1-4 were increased 10-fold compared with the parent antagonist, [desHis(1), Glu(9)]glucagon amide. These potent glucagon antagonists have among the highest pA(2) values of any glucagon analogs reported to date.     

A divalent recombinant immunotoxin formed by a disulfide bond between the extension peptide chains.

Recombinant immunotoxin for the treatment of cancer was made by connecting toxins to 'carcinoma-specific' antibodies that selectively bind to cancer cells, then kills them without harming the normal cells. The divalent recombinant immunotoxin, [B3(Fab)-ext-PE38]2, is a derivative of B3(Fab)-PE38. B3(Fab)-PE38 was made by fusing the Fab domain of the monoclonal antibody (MAb) B3 to PE38, a truncated mutant form of Pseudomonas exotoxin (PE). In this study, B3(Fab)-ext-PE38 was constructed, which has the hinge region of the B3(Fab)-PE38 extended with the peptide extension, G4C(G4S)2, and connected to the C3 connector. The Cys residue of the extension peptide chain makes the disulfide bond between the two Fab domains. The extension sequence (ext) makes the dimerization of B3(Fab)-ext-PE38 easier to form the divalent immunotoxin, because it decreases the steric hindrance between the two PE38s. The constructed genes were expressed in E. coli as inclusion bodies. Polypeptides that were obtained from the inclusion body were refolded, and the active forms were purified. The ID50 values of the divalent molecule, [B3(Fab)-ext-PE38]2, were about 4 ng/ml on A431 cell lines, about 1 ng/ml on CRL1739 cell lines, and 5 ng/ml on MCF-7 cell lines. The [B3(Fab)-ext-PE38]2 showed about a 12-fold higher cytotoxicity on CRL1739 cell lines than B3(scFv)-PE40 did.     

The genome of Oscheius tipulae: determination of size, complexity, and structure by DNA reassociation using fluorescent dye.

This work describes the physicochemical characterization of the genome and telomere structure from the nematode Oscheius tipulae CEW1. Oscheius tipulae is a free-living nematode belonging to the family Rhabditidae and has been used as a model system for comparative genetic studies. A new protocol that combines fluorescent detection of double-stranded DNA and S1 nuclease was used to determine the genome size of O. tipulae as 100.8 Mb (approximately 0.1 pg DNA/haploid nucleus). The genome of this nematode is made up of 83.4% unique copy sequences, 9.4% intermediate repetitive sequences, and 7.2% highly repetitive sequences, suggesting that its structure is similar to those of other nematodes of the genus Caenorhabditis. We also showed that O. tipulae has the same telomere repeats already found in Caenorhabditis elegans at the ends and in internal regions of the chromosomes. Using a cassette-ligation-mediated PCR protocol we were able to obtain 5 different putative subtelomeric sequences of O. tipulae, which show no similarity to C. elegans or C. briggsae subtelomeric regions. DAPI staining of hermaphrodite gonad cells show that, as detected in C. elegans and other rhabditids, O. tipulae have a haploid complement of 6 chromosomes.     

SIMPLE METHOD FOR RNA PREPARATION FROM CYANOBACTERIA1

A simple and rapid method is presented for the preparation of RNA from various cyanobacteria. Unlike other methods that require a lysis solution, lysozymes, or proteinase K, the proposed method, called the bead–phenol–chloroform (BPC) method, uses silica/zirconia beads, phenol, and chloroform to break the cells and extract RNA more efficiently. Experiments confirm that the BPC method can successfully isolate total RNA from various cyanobacterial strains without DNA contamination, and the extracted RNA samples have a relatively high purity, concentration, and yield. Furthermore, the BPC method is more rapid, simple, and economical when compared with previously reported methods.     

Degradations of human immunoglobulins and hemoglobin by a 60 kDa cysteine proteinase of Trichomonas vaginalis

The present study was undertaken to investigate the role of cysteine proteinase of Trichomonas vaginalis in escaping from host defense mechanism. A cysteine proteinase of T. vaginalis was purified by affinity chromatography and gel filtration. Optimum pH for the purified proteinase activity was 6.0. The proteinase was inhibited by cysteine and serine proteinase inhibitors such as E-64, NEM, IAA, leupeptin, TPCK and TLCK, and also by Hg²⁺, but not affected by serine-, metallo-, and aspartic proteinase inhibitors such as PMSF, EDTA and pepstatin A. However, it was activated by the cysteine proteinase activator, DTT. The molecular weight of a purified proteinase was 62 kDa on gel filtration and 60 kDa on SDS-PAGE. Interestingly, the purified proteinase was able to degrade serum IgA, secretory IgA, and serum IgG in time- and dose-dependent manners. In addition, the enzyme also degraded hemoglobin in a dose-dependent manner. These results suggest that the acidic cysteine proteinase of T. vaginalis may play a dual role for parasite survival in conferring escape from host humoral defense by degradation of immunoglobulins, and in supplying nutrients to parasites by degradation of hemoglobin.     

In vitro modeling of liver fibrosis with 3D co-culture system using a novel human hepatic stellate cell line

Hepatic stellate cells (HSCs) play an important role in liver fibrosis; however, owing to the heterogeneity and limited supply of primary HSCs, the development of in vitro liver fibrosis models has been impeded. In this study, we established and characterized a novel human HSC line (LSC-1), and applied it to various types of three-dimensional (3D) co-culture systems with differentiated HepaRG cells. Furthermore, we compared LSC-1 with a commercially available HSC line on conventional monolayer culture. LSC-1 exhibited an overall upregulation of the expression of fibrogenic genes along with increased levels of matrix and adhesion proteins, suggesting a myofibroblast-like or transdifferentiated state. However, activated states reverted to a quiescent-like phenotype when cultured in different 3D culture formats with a relatively soft microenvironment. Additionally, LSC-1 exerted an overall positive effect on co-cultured differentiated HepaRG, which significantly increased hepatic functionality upon long-term cultivation compared with that achieved with other HSC line. In 3D spheroid culture, LSC-1 exhibited enhanced responsiveness to transforming growth factor beta 1 exposure that is caused by a different matrix-related protein expression mechanism. Therefore, the LSC-1 line developed in this study provides a reliable candidate model that can be used to address unmet needs, such as development of antifibrotic therapies.     

Mechanism of modulation of dopamine beta-monooxygenase by pH and fumarate as deduced from initial rate and primary deuterium isotope effect studies

Dopamine beta-monooxygenase catalyzes a reaction in which 2 mol of protons are consumed for each turnover of substrate. Studies of the pH dependence of initial rate parameters (Vmax and Vmax/Km) and their primary hydrogen isotope effects show that at least two ionizable residues are involved in catalysis. One residue (B1, pK = 5.6-5.8) must be protonated prior to the carbon-hydrogen bond cleavage step, implying a role for general-acid catalysis in substrate activation. A second protonated residue (B2, pK = 5.2-5.4) facilitates, but is not required for, product release. Recent measurement of the intrinsic isotope effect for dopamine beta-monoxygenase [Miller, S. M., & Klinman, J. P. (1983) Biochemistry (preceding paper in this issue)] allows an analysis of the pH dependence of rate constant ratios and in selected instances individual rate constants. We demonstrate large changes in the rate-determining step as well as an unprecedented inversion in the kinetic order of substrate release from ternary complex over an interval of 2 pH units. Previously, fumarate has been used in dopamine beta-monooxygenase assays because of its property of enzyme activation. Studies of the pH behavior in the presence of saturating concentrations of fumarate have shown two causes of the activation: (i) fumarate perturbs the pK of B1 to pK = 6.6-6.8 such that the residue remains protonated and the enzyme optimally active over a wider pH range; (ii) fumarate decreases the rate of dopamine release from the ternary enzyme-substrate complex, increasing the equilibrium association constant for dopamine binding. Both effects are consistent with a simple electrostatic stabilization of bound cationic charges by the dianionic form of fumarate.     

Aging and monoamine receptors in brain.

Biochemical evidence is presented for selective decreases in biogenic amine receptor systems with age in the rabbit. Dopamine-stimulated adenylate cyclase activity in striatum, hypothalamus, frontal cortex, and anterior limbic cortex declined by about 50% as rabbits aged from less than 1 to 5 years of age. Similar decreases were found for histamine-stimulated activity in hypothalamus and the cortical regions. These changes were in maximal response rather than in affinity for amine. In contrast, dopamine-stimulated adenylate cyclase of retina and both basal and Gpp(NH)p-stimulated activity in these regions were not altered with age. In addition, with age the number of binding sites for [3H]spiroperidol, a dopamine antagonist, decreased by 30--40% without change in ligand affinity in striatum and limbic cortex. These changes in striatum and cortex occurred in the absence of decreases in either dopamine concentration or choline acetylase activity. It is proposed that selective age-dependent decreases in the functional number of biogenic amine receptors occur in the absence of, or independent from neuronal cell loss, possibly by a mechanism of desensitization. These changes occurred in brain regions that in man are thought to be of importance in the age-related loss of cerebral function.     

Electrochemical treatment of mouse and rat fibrosarcomas with direct current

Electrochemical treatment (ECT) of cancer utilizes direct current to produce chemical changes in tumors. ECT has been suggested as an effective alternative local cancer therapy. However, a methodology is not established, and mechanisms are not well studied. In vivo studies were conducted to evaluate the effectiveness of ECT on animal tumor models. Radiation-induced fibrosarcomas were implanted subcutaneously in 157 female C3H/HeJ mice. Larger rat fibrosarcomas were implanted on 34 female Fisher 344 rats. When the spheroidal tumors reached 10 mm in the mice, two to five platinum electrodes were inserted into the tumors at various spacings and orientations. Ten rats in a pilot group were treated when their ellipsoidal tumors were about 25 mm long; electrode insertion was similar to the later part of the mouse study, i.e., two at the base and two at the center. A second group of 24 rats was treated with six or seven electrodes when their tumors were about 20 mm long; all electrodes were inserted at the tumor base. Of the 24 rats, 12 of these were treated once, 10 were treated twice, and 2 were treated thrice. All treated tumors showed necrosis and regression for both mice and rats; however, later tumor recurrence reduced long-term survival. When multiple treatments were implemented, the best 3 month mouse tumor cure rate was 59.3%, and the best 6 month rat tumor cure rate was 75.0%. These preliminary results indicate that ECT is effective on the radiation-induced fibrosarcoma (RIF-1) mouse tumor and rat fibrosarcoma. The effectiveness is dependent on electrode placement and dosage. Bioelectromagnetics 18:14–24, 1997. © 1997 Wiley-Liss, Inc.