Enzymatic catalysis in organic solvents: Polyethylene glycol modified hydrogenase retains sulfhydrogenase activity in toluene

Naturally occurring enzymes may be modified by covalently attaching hydrophobic groups that render the enzyme soluble and active in organic solvents, and have the potential to greatly expand applications of enzymatic catalysis. The reduction of elemental sulfur to hydrogen sulfide by a hydrogenase isolated from Pyrococcus furiosus has been investigated as a model system for organic biocatalysis. While the native hydrogenase catalyzed the reduction of sulfur to H(2)S in aqueous solution, no activity was observed when the aqueous solvent was replaced with anhydrous toluene. Hydrogenase modified with PEG p-nitrophenyl carbonate demonstrated its native biocatalytic ability in toluene when the reducing dye, benzyl viologen, was also present. Neither benzyl viologen nor PEG p-nitrophenyl carbonate alone demonstrated reducing capability. PEG modified cellulase and benzyl viologen were also incapable of reducing sulfur to H(2)S, indicating that the enzyme itself, and not the modification procedure, is responsible for the conversion in the nonpolar organic solvent. Sulfide production in toluene was tenfold higher than that produced in an aqueous system with equal enzyme activity, demonstrating the advantages of organic biocatalysis. Applications of bio-processing in nonaqueous media are expected to provide significant advances in the areas of fossil fuels, renewable feedstocks, organic synthesis, and environmental control technology. (c) 1996 John Wiley & Sons, Inc.     

γ-Aminobutyric acid (GABA) and other amino acids in tissues of the tick,Amblyomma hebraeum (Acari: Ixodidae) throughout the feeding and reproductive periods

We assayed a variety of tick (Amblyomma hebraeum Koch; Acari, Ixodidae) tissues for a number of amino acids throughout the feeding and early reproductive periods. Our HPLC assay could detect as little as 2–5 pmol per sample of the following: GABA, glycine, serine, glutamine, alanine, taurine, glutamate and aspartate. All of these amino acids could be detected in the salivary gland, synganglion (=total CNS in acarines), haemolymph, Gené's organ, seminal receptacle and ovary. GABA reached high levels in the salivary gland of freshly engorged ticks (685 nmol g^-1) and in the synganglion it exceeded 1000 nmol g^-1 throughout most of the feeding cycle and the first week post-engorgement. GABA also reached a peak titre in the haemolymph of 40 nmol ml^-1. Taurine levels peaked at 1065 nmol g^-1 in the salivary gland from large partially fed ticks. Glutamate and aspartate were likewise found in the salivary gland and synganglion at high concentrations. For most of the amino acids there is insufficient information to correlate these titres (and fluctuations of titres) to neuromodulatory functions. It is possible, however, that the high GABA titre in the salivary gland of engorged ticks is correlated with an augmented level of fluid secretion.Deceased: Department of Pharmacology, University of Alberta, Edmonton, Alberta, Canada, T6G 2E9.     

High-yield production of diphtheria toxin mutants by high-density culture of C7(β)tox+ strains grown in a non-deferrated medium

A high-density growth approach was utilized to produce mutated diphtheria toxin from two strains of Corynebacterium diphtheria: C7 ()^{(tox-201, tox-9)} and C7 ()^(tox-107). The cross-reacting mutants (CRM) of the diphtheria toxin are CRM9 and CRM107; both of them carry the mutation in their binding site and, as a result, have 1/300 of the systemic toxicity of the wild-type diphtheria toxin. Since iron inhibits diphtheria toxin production, the traditional approach has been to grow the bacteria in a very low iron concentration. The procedure described here involved the use of a modified, non-deferrated, growth medium that provided fast and high-density growth of the bacteria, and which, when associated with simultaneous depletion of glucose and iron, enhanced the toxin production. Oxygen-enriched air was supplied to enable the bacteria to grow to a cell density giving an absorbance of 70 at 600 nm (15–20 g/l dry weight). The maximum toxin concentration in the culture supernatant was 150 mg/l. The CRM products, which remained stable following microfiltration and ultrafiltration, could be easily purified using a two-step chromatography procedure.     

Response to Gravity by Zea mays Seedlings : I. Time Course of the Response

Gravistimulation induces an asymmetric distribution of free indole-3-acetic acid (IAA) in the cortex-epidermis of the Zea mays L. cv `Stowells Evergreen' mesocotyl within 15 minutes, the shortest time tested. IAA was measured by an isotope dilution method as the pentaflurobenzyl ester. The per cent IAA in the lower half of the mesocotyl cortex was 56 to 57% at 15, 30, and 90 minutes after stimulus initiation. Curvature is detectable in the mesocotyl within 3 minutes after beginning gravitropic stimulation. The rate of curvature of the mesocotyl increases during the first 60 minutes to a maximum of about 30° per hour. Thus, the growth asymmetry continues to increase for 45 minutes after hormone asymmetry is established.

Free IAA occurs predominantly in the stele of the mesocotyl whereas esterified IAA is mainly in the mesocotyl cortex-epidermis. This compartmentation may permit determining in which tissue the hormone asymmetry arises. Current data suggest the asymmetry originated in the stele.

     

Use and value of serologic tests for the diagnosis of systemic candidiasis in cancer patients: A prospective study of 146 patients

Sera from 146 cancer patients at risk for disseminated candidiasis were studied prospectively with immunodiffusion (ID), counterelectrophoresis (CEP), and latex agglutination (LA) tests to determine their diagnostic value in the detection of antibodies to theCandida species. Serial serum samples, cultures, and clinical data were obtained after a malignancy was diagnosed. Patients were classified into three groups (I, II, and III) on the basis of cultural, histological, and clinical evidence for superficial (Group I) versus disseminated (Group III)Candida infection. Thirty-two of 78 patients (41%) in Group I had positive ID, CEP, and LA titers. In Group II, those patients lacking histological confirmation of disseminated infection, 16 of 18 (89%) had positive titers. Thirty-six of 50 (72%) in Group III were positive by all three tests. Heavy colonization of the gastrointestinal tract, without evidence of tissue invasion, produced positive test results. Negative serologic tests were encountered in immunosuppressed patients with rapidly progressive candidiasis.C. krusei infections produced specific antibody titers detected by the homologous antigen but not byC. albicans antigen. Stable or decreasing LA titers were correlated with clinical improvement in patients receiving effective antifungal therapy.