Enhanced shikonin production from Lithospermum erythrorhizon by in situ extraction and calcium alginate immobilization

Plant cell cultures of Lithospermum erythrorhizon were carried out to produce shikonin by in situ extraction and cell immobilization in calcium alginate bead in shake flask cultures. In situ product extraction and cell immobilization enhanced shikonin production and facilitated product recovery. In situ extraction by n-hexadecane and cell immobilization by calcium alginate gave higher specific shikonin productivities of 7.4 and 2.5 times, respectively, than those from the cultures of free cells without extraction. Simultaneous use of both techniques increased specific and volumetric productivities of shikonin 25- and 15-fold, respectively. In calcium alginate immobilized cell cultures, n-hexadecane addition at an early stage (before 15 days) was effective for shikonin production, and solvent addition after 15 days of the culture significantly reduced shikonin production. Higher numbers of plant cell immobilized bead inoculation did not increase shikonin production and sucrose consumption. Most of the produced shikonin was dissolved in the solvent layer.     

Glucose oxidase as the antifungal principle of talaron from Talaromyces flavus

Analysis of an authentic sample of the antifungal antibiotic talaron from the biocontrol fungus Talaromyces flavus indicated that approximately 40% of the solid sample was glucose oxidase. High-performance liquid chromatography elution profiles of the antimicrobial activity of talaron coeluted with those of glucose oxidase. Fluorescence emission and excitation wavelength maxima for talaron were similar to those of glucose oxidase from Aspergillus niger. The molecular weight of talaron was 152,000 with a subunit molecular weight of 71,000. The isoelectric point of talaron was pH 4.2. Mobilities of talaron on native, sodium dodecylsulfate, and isoelectric focusing polyacrylamide gels were identical with those of glucose oxidase produced by T. flavus. Furthermore, talaron had antimicrobial activity only in the presence of glucose. Hydrogen peroxide produced by the action of glucose oxidase is toxic to Verticillium dahliae. This study indicates that the antifungal activity of authentic talaron resulted from glucose oxidase produced by T. flavus.     

N-bromoacetyl-D-leucylglycine. An affinity label for neutral endopeptidase 24.11

Neutral endopeptidase 24.11 is rapidly inactivated by N-bromoacetyl-D-leucylglycine in a reaction which follows first-order kinetics at pH 8 and 37 degrees C. The concentration dependence of inactivation revealed saturation kinetics with an apparent Ki of 10 mM and kappa inact of 0.4 min-1 at saturating inhibitor concentration. Enzyme can be protected from inactivation by either the substrate Leu5-enkephalin or the competitive inhibitors Phe-Gly or Phe-Ala. Inactivation of enzyme by N-bromo-[14C]acetyl-D-leucylglycine proceeds with the incorporation of a stoichiometric amount of labeled inhibitor. Tryptic digestion of the radioactively labeled enzyme followed by high performance liquid chromatography allowed the isolation of a modified peptide with the sequence T-D-V-H-S-P-G-N-F-R in which histidine (His704) is the modified residue. Site-directed mutagenesis was used to generate a mutant form of the enzyme in which histidine 704 was converted to a glutamine residue. This mutant enzyme retained less than 0.1% of the activity of the native enzyme. These results demonstrate that His704 is at the active site of neutral endopeptidase 24.11 and suggest a catalytic role for this residue.     

AN OVERVIEW OF THE GENUS PYRRHOPAPPUS (ASTERACEAE: LACTUCEAE) WITH EMPHASIS ON CHLOROPLAST DNA RESTRICTION SITE DATA

The genus Pyrrhopappus in recent systematic treatments has comprised five taxa (four species, one with two varieties), which have now been studied anew using morphogeographical and chloroplast DNA restriction site data. Eight populations, representing all of the recognized taxa of Pyrrhopappus, were digested with 17 restriction enzymes. Only three restriction site differences were found from among 750 restriction sites and no length variations were observed. This contrasts with similar studies, using these same enzymes, on the closely related genus Krigia in which 173 mutation sites and 20 length variations were found among the seven species concerned. Nucleotide sequence divergence values among the species of Pyrrhopappus were extremely low (0.0012) compared to much higher values found in the closely related genus Krigia (0.1270). Three species of Pyrrhopappus are herein recognized: two diploids with 2n = 12 chromosomes, P. carolinianus and P. pauciflorus (including P. multicaulis, P. geiseri and P. rothrockii), and a tetraploid (2n = 24), P. grandiflorus. The tetraploid is partially sympatric with both diploids but is readily recognized by its perennial roots, which bear tuber-like enlargements. These three species presumably arose relatively recently, and the DNA data suggest that neither P. pauciflorus nor P. carolinianus gave rise to the tetraploid P. grandiflorus.     

Biological activity of the antitumor protein neocarzinostatin coupled to a monoclonal antibody by N-succinimidyl 3-(2-pyridyldithio)-propionate

The chromophore free apoprotein of neocarzinostatin was coupled to monoclonal IgG₁ antibody using N-Succinimidyl 3-(2-pyridyldithio)-propionate as heterobifunctional reagent. After coupling active chromophore was reassociated with the apoprotein. We present here experimental evidence that the hybrid protein retains biological activity as measured by the degradation of T2-DNA and bacteriostatic action.     

Role of catecholamines in hypotensive response to dieting.

The effect of dieting on blood pressure and catecholamine metabolism was assessed in 11 normotensive obese women by providing first a weight-maintenance regimen high in carbohydrate and then a low-energy diet. All dietary constituents other than carbohydrate were maintained constant throughout the 18-day study. The low-carbohydrate diet led within 48 hours to a 41% fall in the urinary output of 4-hydroxy-3-methoxy mandelate and a significant fall in systolic and diastolic blood pressure. Plasma noradrenaline concentrations also fell and the hypotensive effect of the diet continued despite a maintained total body sodium. Thus the fall in blood pressure appeared to be mediated by changes in catecholamine metabolism independent of sodium intake. This may explain both the usefulness of weight reduction in hypertensive patients and the fainting that occurs in some normotensive obese subjects taking slimming regimens low in carbohydrate.     

Electrophoretic studies of several hydrolytic enzymes in relation to the cold tolerance of alfalfa.

Two cultivars of alfalfa (Medicago sativa L.), cold-tolerant Vernal and cold-sensitive Sonora, were grown under summer, winter, and dehardening environments to determine the characteristics and relationships of several hydrolytic enzymes to cold tolerance.Soluble enzymatic proteins, extracted from lyophilized crown and root tissues with three different solvents, were separated by polyacrylamide disc-gel electrophoresis and evaluated on the basis of equal dry weights of tissue and equal quantities of protein.Gels assayed for amylases, acid phosphatases, esterases, leucine aminopeptidases, and adenosine triphosphatases exhibited mainly quantitative differences in isoenzymes depending upon extractant, cultivar, and environmental differences. The qualitative differences detected were generally due to differential solubilities of isoenzymes in the three extractants and, to a lesser extent, were related to environmental, cultivar, or stability differences.While activities of esterases, acid phosphatases, and leucine aminopeptidases increased in winter samples, as soluble protein increased, only slight decreases in these enzymes occurred during dehardening. Conversely, activities of amylases were slightly lower in winter samples than in the other samples, and adenosine triphosphatase activity decreased in the most coldtolerant sample.The measured levels of total nonstructural carbohydrate, total soluble sugar, and starch indicated differences between cultivars in starch-sugar conversion. Further, the differential heat stabilities of the isoamylases also provided some information as to the nature of “protected activity” of diastatic enzymes.Differential cryostabilities of peptidases and adenosine triphosphatases detected between cultivars and environments also demonstrated the influence of the extraction medium in maintaining enzyme activity, and these observations may be important to an understanding of cold tolerance in alfalfa. The obvious speculations regarding enzyme stability and the factors involved as related to the cold tolerance of alfalfa require further examination.     

Sterically hindered disulfide bridges in cystine diketopiperazine, cysteinyl-cysteine disulfide and derivatives.

L-Cystine diketopiperazine (1), L-cysteinyl-cysteine disulfide -HCl (2), L-cysteinyl-cysteine disulfide methyl ester -HCl (3), and t-butyloxycarbonyl-L-cysteinyl-cysteine disulfide methyl ester (4) are investigated by CD, ultraviolet, 13C NMR, infrared and laser Raman spectroscopy. The temperature dependence of the 13C NMR signals of 1 reveals an exceptionally high energy barrier of deltaGNo. = 15.8 +/- 0.2 kcal/mol for the reversible change in helicity of the inherently dissymmetric disulfide bridge of 1. The P-helical diastereomer predominates in dimethyl-sulfoxide at 25 degrees C, with 80-85% of the molecules having this configuration. The Cotton effects of 1 are larger and show smaller temperature coefficients than the conformationally more mobile cystine compounds 2 and 3. After dissolving crystals of 1 in 95% ethanol there is a time-dependent decrease of the ellipticity of the negative Cotton effect at 225 nm, indicating a conformational change in going from crystal to solution. Besides 1, 2 and 3 are at present the only known examples of cystine derivatives with C-S-S-C torsional angles around 90 degrees, which do not exhibit optical activity in the long wavelength disulfide absorption, as is predicted for 1 from the Linderberg-Michl model. At 305 nm a new weak Cotton effect was discovered for 1. The solvent dependence of the CD spectra is discussed and the infrared and Raman spectra are assigned.