Production of bacteriolytic enzymes by mycoparasitic Trichoderma strains

Eighteen mycoparasitic Trichoderma strains were tested for their ability to degrade heat-inactivated Bacillus cereus var. mycoides, B. megaterium, B. subtilis, Escherichia coli, Micrococcus luteus, Pseudomonas aeruginosa and Serratia marcescens cells. The non-inductive and inductive ferment broths of five strains with good degrading abilities towards B. subtilis were investigated for specific degrading enzyme activities. In addition to trypsin- and chymotrypsin-like protease activities, -1,4-N-acetyl-glucosaminidase (NAGase) was also secreted. Strain Trichoderma harzianum T19 had the most outstanding degrading abilities. The extracellular degrading enzymes of this strain were separated on a Sephadex G-150 column, and their preliminary characterization was performed. The results demonstrated that muramidase-like activities are present in the ferment broth of this T. harzianum strain.     

Malate dehydrogenases--structure and function

Malate dehydrogenases (MDH, L-malate:NAD oxidoreductase, EC 1.1.1.37), catalyze the NAD/NADH-dependent interconversion of the substrates malate and oxaloacetate. This reaction plays a key part in the malate/aspartate shuttle across the mitochondrial membrane, and in the tricarboxylic acid cycle within the mitochondrial matrix. They are homodimeric molecules in most organisms, including all eukaryots and the most bacterial species. The enzymes share a common catalytic mechanism and their kinetic properties are similar, which demonstrates a high degree of structural similarity. The three-dimensional structures and elements essential for catalysis are conserved between mitochondrial and cytoplasmic forms of MDH in eukaryotic cells even though these isoenzymes are only marginally related at the level of primary structure.     

Role of conformational flexibility for enzymatic activity in NADH oxidase from Thermus thermophilus.

NADH oxidase from Thermus thermophilus is a homodimer with an unknown physiological function. As is typical for an enzyme isolated from a thermophile, the catalytic rate, kcat, is low at low temperatures and increases with temperature, achieving an optimum at the physiological temperature of the organism, i.e. at approximately 70 degrees C for T. thermophilus. At low temperatures, the kcat of several enzymes from thermophilic and mesophilic organisms can be increased by chaotropic agents. The catalytic rate of NADH oxidase increases in the presence of urea. At concentrations of 1.0-1.3 m urea it reaches 250% of the activity in the absence of urea, at 20 degrees C. At higher urea concentrations the enzyme activity is inhibited. The urea-dependent activity changes correlate with changes in the fluorescence intensity of Trp47, which is located in the active site of the enzyme. Both fluorescence and circular dichroism measurements indicate that the activation by chaotropic agents involves local environmental changes accompanied by increased dynamics in the active site of the enzyme. This is not related to the global structure of NADH oxidase. The presence of an aromatic amino acid interacting with the flavin cofactor is common to numerous flavin-dependent oxidases. A comparison of the crystal structure with the activation thermodynamic parameters, deltaH* and TdeltaS*, obtained from the temperature dependence of kcat, suggests that Trp47 interacts with a water molecule and the isoalloxazine flavin ring. The present investigation suggests a model that explains the role of the homodimeric structure of NADH oxidase.     

Effect of ethanol on certain metabolic pathways in young R/Amsterdam rats

R/Amsterdam rats were offered a 15% aqueous ethanol solution as drinking fluid from delivery via the milk (Group A) or from the weanling (Group B). Ethanol treatment resulted in a significant retardation of growth in both Groups A and B compared to controls (Group C); the changes were more marked in Group A. Female rats responded to ethanol with higher increase of microsomal G6P-ase and mixed function oxygenase activities than males subjected to the same treatment. Hepatic triglyceride, glycogen and protein contents remained unaffected by ethanol. There was no difference in the changes of liver metabolism between Groups A and B either receiving ethanol already from birth via the milk or only after weanling.     

Synthesis of peptide and pseudopeptide amides inhibiting the proliferation of small cell and epithelial types of lung carcinoma cells

Small cell lung cancer (SCLC) cell lines produce and secrete various peptide hormones, e.g. bombesin (BN)/gastrin releasing peptide (GRP) like peptides that are proposed to function as their autocrine growth factors. To inhibit the proliferative effect of these hormones we have synthesized short chain BN[7-14]-analogues replacing the C-terminal peptide bond by a methylene-amino (-CH₂NH-) unit and introducing d -Phe or d -Ser into position 12. As several substance P (SP) analogues were found to inhibit the growth of SCLC cells, some short chain SP-analogues have been synthesized. (Pseudo)octapeptides were synthesized in solution, by fragment condensation using the DCC/HOPfp method. Fragments and SP-analogues were synthesized stepwise using pentafluorophenyl esters. The resistance to hydrolysis of the reduced peptide bond made permitted exact quantification of the Leuψ(CH₂NH)Leu pseudopeptide in hydrolysates. The binding ability of both types of peptides to BN-receptors on Swiss 3T3 mouse fibroblast cells and their antiproliferative effect on NCI-H69 human SCLC cell line have been tested and compared with a short chain SP-antagonist pHOPA-d -Trp-Phe-d -Trp-Leu-Leu-NH₂ ( R ) previously described as a potent inhibitor of SCLC proliferation. While BN-analogues showed weak activity in inhibition of proliferation of SCLC cells, SP-analogues 6 : d -MePhe-d-T rp-Phe-d -Trp-Leuψ(CH₂NH)-Leu-NH₂ and 7 : d -MePhe-d -Trp-Phe-d -Trp-Leu-MPA, in spite of greatly diminished affinity towards the BN-receptor, inhibited SCLC proliferation more effectively than R ( 6 : IC₅₀=2 μm , 7 : IC₅₀=5 μm and R : IC₅₀=10 μm ). Moreover, 6 inhibited the respiratory activity of SK-MES 1 epithelial type of lung carcinoma cells in proliferating but not in the quiescent state, suggesting that the antiproliferative effect of these compounds is not due to simple cytotoxicity. These short chain analogues of SP might be promising candidates as therapeutic agents in the treatment of SCLC. © 1998 European Peptide Society and John Wiley & Sons, Ltd.     

High-performance liquid chromatographic-tandem mass spectrometric method for the determination of clemastine in human plasma

A highly sensitive high-performance liquid chromatographic-tandem mass spectrometric method (HPLC-MS-MS) has been developed to quantitate clemastine in human plasma for the purpose of pharmacokinetic studies. Sample preparation was carried out by liquid-liquid extraction using deuterated clemastine as an internal standard. Chromatographic separation used a C18 reversed phase polymer column giving an extremely fast total run time of 2 min. The method was validated and used for the bioequivalence study of clemastine tablets in healthy male volunteers (n=28). The lower limit of detection proved to be 0.01 ng/ml for clemastine.     

Changing the recognition specificity of a DNA-methyltransferase by in vitro evolution

The gene coding for the SinI DNA-methyltransferase, a modification enzyme able to recognize and methylate the internal cytosine of the GG^A/_TCC sequence, was subjected to in vitro mutagenesis, DNA-shuffling and a strong selection for relaxed GGNCC recognition specificity. As a result of this in vitro evolution experiment, a mutant gene with the required phenotype was selected. The mutant SinI methyltransferase carried five amino acid substitutions. None of these was found in the ‘variable region’ that were thought to be responsible for sequence specificity. Three were located near the N-terminal end, preceding the first conserved structural motif of the enzyme; two were found between conserved motifs VI and VII. A clone engineered to carry out only the latter two replacements (L214S and Y229H) displays relaxed recognition specificity similar to that of the parental mutant, whereas the clone carrying only the N-terminal replacements showed a much weaker change in recognition specificity. The enzyme with two internal mutations was purified and characterized. Its catalytic activity (k_cat/K_m) was ~5-fold lower towards GG^A/_TCC and 20-fold higher towards GG^G/_CCC than that of the wild-type enzyme.     

Opioid receptor binding characteristics and structure-activity studies of novel tetrapeptides in the TIPP (Tyr-Tic-Phe-Phe) series

The development of the prototype synthetic delta-opioid receptor antagonist peptides TIPP [(H-Tyr-Tic-Phe- Phe-OH); Tic: tetrahydroisoquinoline-3-carboxylic acid] and TIPPpsi (H-Tyr-psiTic-Phe-Phe-OH) by Schiller and coworkers was followed by extensive structure-activity relationship studies, leading to the emergence of numerous analogs that are of pharmacological interest. Eight novel diastereomeric compounds in this peptide family were designed, prepared, and tested biologically to gain structure-activity relationship information. The new multisubstituted tetrapeptide analogs contain both a 2',6'-dimethyltyrosine residue at the N-terminus and beta-methyl-cyclohexylalanine at the third position as replacements for the original first tyrosine and the third phenylalanine, respectively. These derivatives wear either free acidic (-COOH) or amidated (-CONH2) C-terminal. The potency and delta- versus mu-opioid receptor selectivity were evaluated by in vitro radioreceptor-binding assays, while the intrinsic G-protein-activating efficacy of these analogs was tested in [35S]GTPgammaS-binding assays using rat brain membranes or Chinese hamster ovary cells stably expressing mu- or delta-opioid receptors. The analogs showed delta-antagonist selectivity with differences regarding their isomeric forms, and these analogs containing a C-terminal carboxamide group displayed a mixed mu-agonist/delta-antagonist profile, thus they are expected to be safer analgesics with a low propensity to produce tolerance and physical dependence. These results constitute further examples of the influence of beta-methyl substitution and C-terminal amidation on potency, selectivity, and signal transduction properties of TIPP-related peptides as well as they represent valuable pharmacological tools for opioid research.     

Microcalorimetric study of elongation factor Tu from Thermus thermophilus in nucleotide-free,GDP and GTP forms and in the presence of elongation factor Ts

Elongation factor (EF) Tu undergoes profound nucleotide-dependent conformational changes in its functional cycle. The thermodynamic parameters of the different Thermus thermophilus EF-Tu forms, its domains I, II/III and III, were determined by microcalorimetry. Thermal transitions of the EF-Tu.GDP and EF-Tu.guanosine-5'-[beta,gamma-imido]triphosphate have a cooperative two-state character. Nucleotide removal affected the cooperativity of the thermal transition of EF-Tu. Microcalorimetric measurements of nucleotide-free EF-Tu and its separated domains showed that domains II/III have the main stabilizing role for the whole protein. Despite the fact that strong interactions between elongation factors Tu and Ts from T. thermophilus at 20 degrees C exist, the thermal transition of neither protein in the complex was significantly affected.