Antibody responses of mice exposed to low-power microwaves under combined, pulse-and-amplitude modulation

Irradiation by pulsed microwaves (9.4 GHz, 1 microsecond pulses at 1,000/s), both with and without concurrent amplitude modulation (AM) by a sinusoid at discrete frequencies between 14 and 41 MHz, was assessed for effects on the immune system of Balb/C mice. The mice were immunized either by sheep red blood cells (SRBC) or by glutaric-anhydride conjugated bovine serum albumin (GA-BSA), then exposed to the microwaves at a low rms power density (30 microW/cm2; whole-body-averaged SAR approximately 0.015 W/kg). Sham exposure or microwave irradiation took place during each of five contiguous days, 10 h/day. The antibody response was evaluated by the plaque-forming cell assay (SRBC experiment) or by the titration of IgM and IgG antibodies (GA-BSA experiment). In the absence of AM, the pulsed field did not greatly alter immune responsiveness. In contrast, exposure to the field under the combined-modulation condition resulted in significant, AM-frequency-dependent augmentation or weakening of immune responses.     

Application of natural and amplification created restriction sites for the diagnosis of PKU mutations.

PCR amplification, either conventional, or as site directed mutagenesis using primers with mismatched 3'-ends, followed by restriction endonuclease digestion, provides rapid, non-isotope assays of known mutations in the human phenylalanine hydroxylase gene. Such assays were shown to have the potential to detect all of the 18 presently reported phenylketonuria mutations. The practical applicability of this approach was demonstrated for eight mutations in Norwegian phenylketonuria patients, among them the most common ones.     

Partial purification and characterization of two sesquiterpene cyclases from sage (Salvia officinalis) which catalyze the respective conversion of farnesyl pyrophosphate to humulene and caryophyllene

Humulene cyclase and caryophyllene cyclase, two enzymes which catalyze the cyclization of farnesyl pyrophosphate to the respective sesquiterpene olefins, have been partially purified from the supernatant fraction of a sage (Salvia officinalis) leaf epidermis extract and separated from each other by a combination of hydrophobic interaction, gel filtration, and ion-exchange chromatography. The molecular weight of both cyclases was estimated by gel filtration to be 57,000 and both cyclases exhibited a pH optimum of 6.5 and preferred Mg2+ (Km approximately 1.5 mM) as the required divalent metal cation. Both enzymes possessed a Km of about 1.7 microM for farnesyl pyrophosphate, were strongly inhibited by p-hydroxymercuribenzoate, and exhibited comparable sensitivities to a variety of other potential inhibitors. The properties of the two sesquiterpene olefin cyclases, which are the first from a higher plant source to be examined in detail, were very similar to each other and to other monoterpene, sesquiterpene, and diterpene cyclases previously described.     

Cloning of Trametes versicolor Genes Induced by Nitrogen Starvation

We have screened a genomic library of Trametes versicolor for genes whose expression is associated with nitrogen starvation, which has been shown to induce ligninolytic activity. Using two different approaches based on differential expression, we isolated 29 clones. These were shown by restriction mapping and cross-hybridization to code for 11 distinct differentially expressed genes. Northern analysis of the kinetics of expression of these genes revealed that at least four of them have kinetics of induction that parallel kinetics of induction of ligninolytic activity.     

Evidence for the ionization steps in monoterpene cyclization reactions using 2-fluorogeranyl and 2-fluorolinalyl pyrophosphates as substrates

Conversion of geranyl pyrophosphate to cyclic monoterpenes is considered to involve the preliminary isomerization of this acyclic precursor to enzyme-bound linalyl pyrophosphate and the cyclization of this tertiary intermediate. 2-Fluorogeranyl pyrophosphate and 2-fluorolinalyl pyrophosphate are effective competitive inhibitors of the cyclization of geranyl pyrophosphate by several different monoterpene cyclases, and the electron withdrawing alpha-fluorine substituent was shown to suppress the rate of cyclic product formation from both tritium-labeled analogs by at least two orders of cyclic These results indicate that both steps of the coupled isomerization-cyclization sequence are initiated by ionization of an allylic pyrophosphate, and they confirm the electrophilic nature of this enzymatic reaction type and its similarity to the prenyltransferase reaction.     

Biosynthesis of monoterpenes. Enantioselectivity in the enzymatic cyclization of (+)- and (-)-linalyl pyrophosphate to (+)- and (-)-bornyl pyrophosphate

Enzymes from Salvia officinalis and Tanacetum vulgare leaf epidermis catalyze the conversion of the acyclic precursor geranyl pyrophosphate to the cyclic monoterpenes (+)- and (-)-bornyl pyrophosphate, respectively. The antipodal cyclizations are considered to proceed by the initial isomerization of the substrate to the respective bound tertiary allylic intermediates (-)-(3R)- and (+)-(3S)-linalyl pyrophosphate. [(3R)-8,9-14C,(3RS)-1E-3H] Linalyl pyrophosphate (3H:14C = 5.22) was tested as a substrate with the cyclases from both sources to determine the configuration of the cyclizing intermediate. This substrate yielded (-)-bornyl pyrophosphate with 3H:14C ratio greater than 31, indicating specific utilization of (+)-(3S)-linalyl pyrophosphate as predicted. With the (+)-bornyl pyrophosphate cyclase, the 3H:14C ratio of the product was about 4.16, indicating a preference for the (-)-(3R)-enantiomer, but the ability also to utilize (+)-(3S)-linalyl pyrophosphate. (3R)- and (3S)-[1Z-3H]Linalyl pyrophosphate were separately compared to the achiral precursors [1-3H] geranyl pyrophosphate and [1-3H]neryl pyrophosphate (cis-isomer) as substrates for the cyclizations. All functional precursors afforded optically pure (-)-(1S,4S)-bornyl pyrophosphate with the T. vulgare-derived cyclase (as determined by chromatographic separation of diastereomeric ketals of the derived ketone camphor), and (+)-(3S)-linalyl pyrophosphate was the preferred substrate. With the (+)-bornyl pyrophosphate cyclase from S. officinalis, geranyl, neryl, and (-)-(3R)-linalyl pyrophosphates gave the expected (+)-(1R,4R)-stereoisomer as the sole product, and (-)-(3R)-linalyl pyrophosphate was the preferred substrate. However, (3S)-linalyl pyrophosphate yielded (-)-(1S,4S)-bornyl pyrophosphate, albeit at lower rates, indicating the ability of this enzyme to catalyze the anomalous enantiomeric cyclization.     

Stereoscopic back-scattered electron imaging of silver-stained proteins in nucleoli

By using simultaneously the AgNOR silver staining method, back-scattered electron imaging mode and stereo-tilt in scanning electron microscopy (SEM), it is possible to observe the nucleus through the cell surface, the nucleolus, and the tri-dimensional distribution of the AgNOR-associated acidic proteins. In C3H10T1:2 cells and their 7-12-dimethylbenz--anthracene-treated transformants, the staining demonstrates several intranucleolar silver-staining granules (SSG), surrounded by a weakly staining region. The SSG may represent the fibrillar center (FC) and the weakly staining region, the fibrillar dense component (FD). This component can link several SSG together to form a rope-like structure. In cells with no visible nucleolus and inactive nucleolar organizer regions (NORs) the silver-staining granules are less numerous, close together and the presumed fibrillar dense components are not visible. The SSG are located more peripheraly, and the weakly staining region and the rope-like structure are less prominent in control cell nucleoli than in transformed cells with a comparatively high rate of RNA synthesis.     

Role of protein synthesis and proteases in production and inactivation of maturation-promoting activity during meiotic maturation of starfish oocytes

In starfish oocytes, activity of the maturation-promoting factor (MPF) and that of a major cAMP-independent protein kinase dropped at the time of meiotic cleavage, and rose again after the first but not the second meiotic cleavage. Protein synthesis was required before the first meiotic cleavage for both MPF and protein kinase activity to rise again after the first meiotic cleavage. Microinjection of either leupeptin or soybean trypsin inhibitor early enough prior to first polar body emission suppressed both the meiotic cleavage and the associated drop of MPF activity. Microinjection of leupeptin or soybean trypsin inhibitor during the 10-min period before the first meiotic cleavage also suppressed cytokinesis but did not prevent a decrease in MPF activity at the normal time of cytokinesis. The lysosomotropic inhibitor ammonia neither suppressed cytokinesis nor the drop of MPF activity at the time of first meiotic cleavage. Activity of neutral proteases sensitive to leupeptin and soybean trypsin inhibitor was demonstrated in oocyte homogenates prepared at the time of first meiotic cleavage. It is proposed that such proteases might be involved in degradation of protein kinase(s) and in the drop of MPF activity at the time of first meiotic cleavage.