On the multiplicity of sugar transport systems in guinea pig jejunum.

A study has been made in everted sacs of guinea pig jejunum to see if the two transport systems of glucose analogues characterized at the brush border membrane vesicles are operative. The transport kinetics of D-galactose and alpha-methylglucoside up to 80 mM concentrations has been studied, as well as the mutual inhibitions between them at low and high concentrations of the substrate and at different concentrations of the inhibitor. Low temperature (20 degrees C) inhibits galactose transport at 0.1 mM (70%) and 40 mM (78%) concentrations. A mass transfer coefficient, KD, somewhat higher for galactose than for alpha-methylglucoside, was obtained when the transport component was abolished by phlorizin. The transport of D-galactose and alpha-methylglucoside seemed to be compatible with the function of one system shared by both substrates, which presents greater affinity for alpha-methylglucoside. The functional existence of two systems of active transport at the brush border of guinea pig was not evidenced in intestinal preparations of whole tissue, due perhaps to the effect of the unstirred water layers. However, differences in KD values and some results of the mutual inhibitions may suggest a second system.     

Surface heterogeneity of rat sperm during maturation

Rat sperm isolated from the caput and caudal epididymis and the vas deferens were subjected to multiple partition in aqueous two-phase systems. The technique was used to reveal heterogeneity of a sperm population with respect to particular surface properties. Sperm from all three regions gave broad distributions indicative of heterogeneous cell populations. Greatest heterogeneity was observed for cauda sperm with caput and was sperm producing similar distributions.Following multiple partition sperm from different regions of the distribution profiles were immunostained with three antibodies known to recognise maturation antigens. The results show that some antigens are acquired during epididymal transit whilst others are present throughout.The partition (surface heterogeneity) seen cannot therefore be explained solely by the distribution of the antigens recognised by 2D6, 6B2 and 3D5.     

Long-lived primary radical pair state detected by time-resolved fluorescence and absorption spectroscopy in an isolated Photosystem two core

A Photosystem two (PS II) core preparation containing the chlorophyll a binding proteins CP 47, CP 43, D1 and D2, and the non-chlorophyll binding cytochrome-b559 and 33 kDA polypeptides, has been isolated from PS II-enriched membranes of peas using the non-ionic detergent heptylthioglucopyranoside and elevated ionic strengths. The primary radical pair state, P680⁺Pheo^-, was studied by time-resolved absorption and fluorescence spectroscopy, under conditions where quinone reduction and water-splitting activities were inhibited. Charge recombination of the primary radical pair in PS II cores was found to have lifetimes of 17.5 ns measured by fluorescence and 21 ns measured by transient decay kinetics under anaerobic conditions. Transient absorption spectroscopy demonstrated that the activity of the particles, based on primary radical pair formation, was in excess of 70% (depending on the choice of kinetic model), while time-resolved fluorescence spectroscopy indicated that the particles were 91% active. These estimates of activity were further supported by steady-state measurements which quantified the amount of photoreducible pheophytin. It is concluded that the PS II core preparation we have isolated is ideal for studying primary radical pair formation and recombination as demonstrated by the correlation of our absorption and fluorescence transient data, which is the first of its kind to be reported in the literature for isolated PS II core complexes from higher plants.Abbreviations CP 43 and CP 47 chlorophyll binding proteins of PS II having apparent molecular weights on SDS-PAGE of 43 kDa and 47 kDa, respectively - D1 and D2 polypeptides PS II reaction centre polypeptides encoded by the psbA and psbD genes, respectively - HPLC high performance liquid chromatography - PS II Photosystem two - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - P680 primary electron donor of PS II - Pheo phenophytin a - SPC single photon counting - PBQ phenyl-p-benzoquinone - DPC 1,5-diphenylcarbazide AFRC Photosynthesis Research Group, Department of Biochemistry     

Oxidation of methane in boreal forest soils: a comparison of seven measures

Methane oxidation rates were measured in boreal forest soils using seven techniques that provide a range of information on soil CH₄ oxidation. These include: (a) short-term static chamber experiments with a free-air (1.7 ppm CH₄) headspace, (b) estimating CH₄ oxidation rates from soil CH₄ distributions and (c)²²²Rn-calibrated flux measurements, (d) day-long static chamber experiments with free-air and amended (+20 to 2000 PPM CH₄) headspaces, (e) jar experiments on soil core sections using free-air and (f) amended (+500 ppm CH₄) headspaces, and (g) jar experiments on core sections involving tracer additions of¹⁴CH₄. Short-term unamended chamber measurements,²²²Rn-calibrated flux measurements, and soil CH₄ distributions show independently that the soils are capable of oxidizing atmospheric CH₄ at rates ranging to < 2 mg m^–2 d^–1. Jar experiments with free-air headspaces and soil CH₄ profiles show that CH₄ oxidation occurs to a soil depth of 60 cm and is maximum in the 10 to 20 cm zone. Jar experiments and chamber measurements with free-air headspaces show that CH₄ oxidation occurs at low (< 0.9 ppm) thresholds. The¹⁴CH₄-amended jar experiments show the distribution of end products of CH₄ oxidation; 60% is transformed to CO₂ and the remainder is incorporated in biomass. Chamber and jar experiments under amended atmospheres show that these soils have a high capacity for CH₄ oxidation and indicate potential CH₄ oxidation rates as high as 867 mg m^–2 d^–1. Methane oxidation in moist soils modulates CH₄ emission and can serve as a negative feedback on atmospheric CH₄ increases.     

Protein secondary structure of the isolated photosystem II reaction center and conformational changes studied by Fourier transform infrared spectroscopy.

The secondary structure of the photosystem II (PSII) reaction center isolated from pea chloroplasts has been characterized by Fourier transform infrared (FTIR) spectroscopy. Spectra were recorded in aqueous buffers containing H2O or D2O; the detergent present for most measurements was dodecyl maltoside. The broad amide I and amide II bands were analyzed by using second-derivative and deconvolution procedures. Absorption bands were assigned to the presence of alpha-helices, beta-sheets, turns, or random structure. Quantitative analysis revealed that this complex contained a high proportion of alpha-helices (67%) and some antiparallel beta-sheets (9%) and turns (11%). An irreversible decrease in the intensity of the band associated with the alpha-helices occurs upon exposure of the isolated PSII reaction center to bright illumination. This loss of alpha-helical content gave rise to an increase in other secondary structures, particularly beta-sheets. After similar pretreatment with light, sodium dodecyl sulfate polyacrylamide gel electrophoresis reveals lower mobility and solubility of constituent D1 and D2 polypeptides of the PSII reaction center. Some degradation of these polypeptides also occurs. In contrast, there is no change in the mobility of the two subunits of cytochrome b559. In the absence of illumination, the PSII reaction center exchanged into dodecyl maltoside shows good thermal stability as compared with samples in Triton X-100. Only at a temperature of about 60 degrees C do spectral changes take place that are indicative of denaturation.     

Members of the alpha-amylase inhibitors family from wheat endosperm are major allergens associated with baker's asthma

We have identified the major antigens or IgE binding components from wheat flour. Thirty-five sera from patients with baker's asthma were used to analyze the reaction with wheat salt-soluble proteins. We found a 15 kDa SDS-PAGE band which reacted with all sera tested. Purified members of the alpha-amylase inhibitor family, which are the main components of the 15 kDa band, were recognized by specific IgE when tested with a pool of reactive sera. Immunodetection after two-dimensional electrophoretic fractionation of crude inhibitor preparations from wheat endosperms also detected several inhibitor subunits as major low-molecular-weight allergens.     

Measurement of oxidation-reduction midpoint potentials by room temperature electron paramagnetic resonance potentiometry

A room temperature electron paramagnetic resonance potentiometric cell has been developed for the measurement of oxidation-reduction midpoint potentials of enzymes containing paramagnetic centers. Based upon an aqueous flat cell designed for use with the Varian TM high sensitivity cavity, the apparatus combines a high degree of anaerobiosis with low volume requirements. The cell is simple in design, easily constructed, and can be adapted for use with most spectrometer cavities. Tests of the cell using xanthine oxidase, in 50 mM Bicine buffer, pH 7.7, yielded midpoint potentials of -345 and -371 mV for the Mo(VI)/Mo(V) and Mo(V)/Mo(IV) couples compared with values of -373 and -377 mV obtained by electron paramagnetic resonance analysis of frozen potentiometric samples. These values indicate that shifts, of the order of 20-40 mV, may occur upon freezing poised samples. For the Mo center of xanthine oxidase, these shifts in potential are more pronounced for the Mo(VI)/Mo(V) couple and result in a destabilization of the Mo(V) intermediate during freezing.     

Monoamine containing varicosities in the neural sheath of a gastropod mollusc demonstrated by glyoxylic acid histofluorescence

Summary The glyoxylic acid monoamine-detecting technique of Bolstad et al. (1979) was applied to wholemounts of intact ganglia of the gastropod mollusc Philine aperta. A dense network of fluorescent varicosities was found in the sheath of the suboesophageal, visceral and genital ganglia, and most of their nerves. Drug treatment experiments suggest that these projections to the neural sheath contain serotonin. The significance of these terminals in the sheath is discussed and proposals for further work are given.     

Independent control of sexual and scent marking behaviors of male gerbils by cells in or near the medial preoptic area

This research studied the role of the medial preoptic area and adjacent cell populations in androgen control of scent marking and sexual behavior in male gerbils (Meriones unguiculatus). Experiment 1 replicated previous research showing that implants of testosterone propionate in or near the medial preoptic area reinstate marking behavior in castrates. Implant sites near the diagonal band of Broca or in the posterior part of the medial preoptic area, near the anterior hypothalamus, are more effective than other sites. Experiment 2 showed that medial preoptic area lesions permanently impair sexual behavior despite testosterone stimulation. Experiments 2–4 showed that lesions in or near the medial preoptic area can also disrupt scent marking; however, this behavior gradually recovered in many lesioned males, especially if they received testosterone. The data suggest that both scent marking and sexual behavior are controlled by androgens acting on cells in or near the medial preoptic area, but the cell populations involved in these two behaviors are probably not the same.     

Polysaccharides and their common proteinic carrier in the Vi antigens of Citrobacter ballerup and Salmonella typhi Ty2

Immunochemical analysis of Citrobacter ballerup and Salmonella typhi Ty2 showed that the strains share native and heat-resistant proteins that are, apparently, the carriers of a common polysaccharidic determinant present in their respective somatic antigens. After the classic acetic acid hydrolysis, the somatic antigen of C. ballerup reacted, in agar gel, against the homologous antiserum by two precipitation lines, one of which also precipitated against the anti S, typhi Ty2 serum; the hydrolysis of the S. typhi Ty2 somatic antigen demonstrated that, in addition to the 'O' polysaccharide, reacting against all the S. typhi antisera, it contains a polysaccharide that precipitated against the anti-C. ballerup serum. The elusiveness in the agglutinability of only freshly isolated bacterial authorizes some doubt concerning the responsibility of the antipolysaccharide antibodies in the agglutinating Vi sera; in order to induce anitpolysaccharides hyperimmunizations are needed while antiproteins are easily induced by short immunizations.