Additive and independent responses in a single receptor: aspartate and maltose stimuli on the tar protein

The aspartate and maltose responses of E. coli are mediated through a single membrane receptor, yet the responses are independent and additive. Both stimuli cause methylation of the same 4 glutamic acid residues. More extensive methylation occurs when a cell that has adapted to one stimulus is exposed to the second, or when both stimuli are added simultaneously. The degree of methylation, as well as receptor migration on two-dimensional gels, demonstrates that only one type of protein is involved, rather than two different receptors arising from differential processing of a single gene. A conformational "push-pull" mechanism in which binding of stimulus and covalent modification, producing opposing stresses, can explain these diverse results.     

Evaluation of the single radial-immunodiffusion assay for measuring the glycoprotein content of rabies vaccines

The glycoprotein content of rabies vaccines containing the Pitman-Moore strain of rabies virus was measured by the single radial immunodiffusion assay and correlated with vaccine potency. The variability of this assay was 6.3% for a single vaccine lot tested over a one-year period. Using sera prepared against rabies virus glycoprotein from different strains of virus, the assay gave different values. These differences could be eliminated by using a homologous vaccine strain as an internal reference. Single radial-immunodiffusion values for Pitman-Moore vaccines correlated with the manufacturers' NIH potency assay, but required a mathematical transformation to convert values from one assay to the other. Single radial-immunodiffusion values for Street Alabama Dufferin and Flury-LEP vaccines did not correlate with NIH values. Modification of the single radial immunodiffusion technique and the feasibility of using this assay for the determination of rabies vaccine potency are discussed.     

Short- and long-term effects of proallatotoxin (ethoxyprecocene II) on Rhodnius prolixus females

Oogenesis and oviposition can be inhibited in female of Rhodnius prolixus by means of short-term experiment (first reproductive cycle) of a single dose of ethoxyprecocene II given by ingestion. The inhibition is dose-dependent as measured by oocyte growth, egg maturation and egg deposition. In a long-term experiment (second and third reproductive cycles) egg production and oogenesis can be partially or totally re-established by subsequent blood meals without ethoxyprecocene II. These findings suggest that in female R. prolixus, damage caused to corpus allatum by ethoxyprecocene II, in certain cases, is not irreversible.     

Preparation of scirpentriol and triacetoxyscirpenol in good yield from cultures of Fusarium sambucinum NRRL 13495

Crude extracts of filtrates of cultures of Fusarium sambucinum NRRL 13495 were acetylated or hydrolyzed. After chromatography on cartridge columns of silica gel and recrystallization three times from mixtures of ethyl acetate and hexane, 3,4,15-triacetoxyscirpenol (435 +/- 10 mg/liter of filtrate; mean +/- standard error [n = 3]) and the parent alcohol scirpentriol were isolated (261 +/- 29 mg/liter of filtrate; mean +/- standard error [n = 3]) in 68 and 53% yield for a 130- and 14-fold improvement, respectively, over prior reports.     

Construction of DNA-probes and their immunochemical detection using nonradioactive hybridization tests

Simple and efficient chemical approaches to preparation of DNA probes carrying 2,4-dinitrophenyl, dansyl or biotin residues were developed. The residues were introduced using following DNA derivatization procedures: a) transamination of cytidine residues with O-(4-aminobutyl)hydroxylamine; b) mercuration of pyrimidine residues followed by beta-mercaptoethanol modification. It was shown that 2,4-dinitrophenyl-containing DNA probes can be used for nonradioactive hybridization detection of nucleic acids. DNP-DNA: DNA complexes were detected using mouse antibodies specific to 2,4-dinitrophenyl groups, which were developed with peroxidase-conjugated antimouse immunoglobulins. Peroxidase-catalyzed chemoluminescent reaction of luminol oxidation with hydrogen peroxide allowed to detect 10 picograms of the dinitrophenylated single-stranded DNA probe.     

Determination of interproton distances in peptides by two-dimensional nuclear Overhauser effect spectroscopy (NOESY). Conformation of a cyclic analog of substance P in a solution

Quantitative method is developed for evaluation interproton distances in peptides in solution. The method is based on the measurement of the relative intensities of the cross-peaks in the pure-phase absorption NOESY spectra. The ratios of the cross-peak intensities IN alpha/I alpha N and INN/I alpha N enable to determine the corresponding interproton distances dN alpha, d alpha N and dNN for several amino acid residues. These distances can be used to estimate other distances with cross-peaks in NOESY spectra. As example, the interproton distances are determined in a cyclic hexapeptide, namely cyclic analogue of substance P: cyclo [H-Glu-Phe-Phe-Gly-Leu-Met-NH(CH2)3-NH-]. The spatial structure of the molecule in dimethylsulphoxide solution is established.     

Pheophytin-mediated energy storage of photosystem II particles detected by photoacoustic spectroscopy

The photoacoustic (PA) characteristics (energy storage and heat dissipation) of photosystem II (PSII) core-enriched particles from barley were studied (i) in conditions where there was electron flow, i.e., in the presence of a combination of the electron acceptor K₃ Fe (CN)₆, referred to as FeCN, and the electron donor diphenylcarbazide (DPC), and (ii) in conditions where electron flow was suppressed, i.e., in the absence of FeCN and DPC. The experimental data show that a decrease of heat dissipation with a minimum at 540 nm can be interpreted as energy storage resulting from the presence of pheophytin (Pheo) in the PSII particles. On account of the capability of the PA method to measure the energy absorbed by the chromophores which is converted to heat, it is suggested that the PA detection of Pheo present in the PSII complex will permit to clarify the function of processes involving non-radiative relaxation of excited states in P680-Pheo-Q_A interactions.Abbreviations -Car -Carotene - Chl Chlorophyll - DPC Diphenylcarbazide - EPR Electron Paramagnetic Resonance - FeCN potassium ferricyanide - HEPES N-2-hydroxyethylenepiperazine-N-2-ethanesulfonate - P680 reaction center of PSII - PA Photoacoustic - Pheo pheophytin - PSI photosystem I - PSII photosystem II - Q_A primary electron acceptor of PSII     

Purification of bovine liver histidyl-tRNA synthetase, the Jo-1 antigen of polymyositis: size of the whole enzyme and its characteristic proteolytic fragments

We have recently reported the marked increase in frequency which can be achieved in the detection of the anti-Jo-1 antibody of polymyositis in serum samples by replacing commercial mixtures of cytoplasmic and nuclear antigens with the purified antigen, histidyl-tRNA synthetase. The present paper describes a method for purifying this antigen and an investigation of its size. Molecular masses previously reported for the enzyme have varied from 85-154 kDa and subunit molecular masses varying from 40-77 kDa have been observed. Several of these fragments are of sizes similar to those of a number of other autoantigens commonly observed in connective tissue diseases. Since the clinical identification of these autoantigens often relies exclusively on size determination by Western blotting, we have characterized the commonly occurring fragments of histidyl-tRNA synthetase lest they confuse such identification. It is concluded that histidyl-tRNA synthetase, like many other aminoacyl-tRNA synthetases, is subject to severe proteolysis during extraction procedures. Several characteristic fragments (Mr = 80, 75, 61, 55, 50 and 45 kDa) result, a finding that provides a satisfactory explanation of the various values previously reported. The intact bovine enzyme is a dimer of molecular mass close to 160 kDa.     

Investigations on circumferential microfilament bundles in rat retinal pigment epithelium

The presence of contractile proteins in normal rat retinal pigment epithelium has been studied using fluorescence and electron microscopy. Investigations using the F-actin binding toxin, phallacidin, coupled to the fluorochrome nitrobenzoxadiazole, revealed a band of fluorescence at or near the cell membrane. Immunofluorescent observations with anti-myosin and anti-alpha-actinin antisera gave similar results. Electron microscopy employing glutaraldehyde-8% tannic acid fixation revealed the presence of a circumferential microfilament band beneath the pigment epithelial apical surface that is closely associated with the plasma membrane and junctional complexes. Freeze-fracture studies confirmed the relationship of this band to the junctional complexes. The microfilament band measures approximately 0.5 micron +/- 0.2 micron in width and is composed of numerous 6 to 7 nm filaments. Some microtubules are seen in regions around the band, but no organelles appear to be associated with this structure. In en face sections through the zonula adherens, the circumferential microfilament band is associated with 30-nm electron-dense particles that are bound to the internal side of the membrane. Morphological evidence suggests that these may serve in anchoring the band to the membrane and assist in aligning the microfilament bands of adjoining cells. In the subapical cytoplasm, a microfilament bundle network was detected that interfaced with the circumferential microfilament band. In some cases, pigment epithelium was incubated in media-199 containing 25 to 50 ng/ml phallacidin prior to fixation. Circumferential microfilament bands of tissues treated in this manner exhibited a striated appearance.