Synthesis of mannose oligosaccharides via reversal of the α-mannosidase reaction

Summary Three naturally occurring isomers of the disaccharideO--d-mannosyl-d-mannoside were synthesized by reversing the hydrolytic activity of jack bean -mannosidase at 75°C in a very high concentration of mannose. Higher oligosaccharides were also obtained at the later stages of the reaction. The maximum total yield of disaccharides was 37% (w/w) based on the total amount of saccharides.     

Li+ counterion self-diffusion in ordered DNA

The anisotropic self-diffusion coefficient of ⁷Li⁺ (I = 3/2) counterions has been studied in hydrated, macroscopically oriented Li-(B)DNA fibers at relatively high water contents, corresponding to approximate DNA-DNA helix axis distances of 22–35 Å, using the pulsed field gradient hmr spin-echo method. Self-diffusion coefficients parallel (D_∥) and perpendicular (D_?) to the DNA helix axis increase with increasing salt content and with increasing DNA-DNA helix axis distance. The observed anisotropy D_∥/D_? decreases from 1.6 to 1.2 with the DNA-DNA separation increasing from 22 to 35 Å in the salt-free sample. This result can be understood by the obstruction effect caused by the DNA molecules themselves. The values of the Li⁺ self-diffusion coefficients in the most water-rich system with no added salt (corresponding to an approximate distance of 35 Å between the DNA helix axes) were D_∥ ～ 1.15 × 10^?10 m² s^?1 and D_? ～ 0.98 × 10^?10 m² s^?1, compared to 9.14 × 10^?10 m² s^?1 for the diffusion of Li⁺ in an aqueous solution of LiCl (～ 2.1M). The possible occurrence of restriction effects in the DNA fibers have also been studied by determining the self-diffusion coefficient at different effective diffusion times. The self-diffusion coefficient of Li⁺ in the sample with the largest DNA-DNA helix axis distance seems to be independent of the effective diffusion time, which indicates that the lithium ions are not trapped within impermeable barriers. The possibility of diffusion through permeable barriers has also been investigated, and is discussed. © 1994 John Wiley & Sons, Inc.     

Photosystem II in a mutant of Chlamydomonas reinhardtii lacking the 23 kDa psbP protein shows increased sensitivity to photoinhibition in the absence of chloride

The psbP gene product, the so called 23 kDa extrinsic protein, is involved in water oxidation carried out by Photosystem II. However, the protein is not absolutely required for water oxidation. Here we have studied Photosystem II mediated electron transfer in a mutant of Chlamydomonas reinhardtii, the FUD 39 mutant, that lacks the psbP protein. When grown in dim light the Photosystem II content in thylakoid membranes of FUD 39 is approximately similar to that in the wild-type. The oxygen evolution is dependent on the presence of chloride as a cofactor, which activates the water oxidation with a dissociation constant of about 4 mM. In the mutant, the oxygen evolution is very sensitive to photoinhibition when assayed at low chloride concentrations while chloride protects against photoinhibition with a dissociation constant of about 5 mM. The photoinhibition is irreversible as oxygen evolution cannot be restored by the addition of chloride to inhibited samples. In addition the inhibition seems to be targeted primarily to the Mn-cluster in Photosystem II as the electron transfer through the remaining part of Photosystem II is photoinhibited with slower kinetics. Thus, this mutant provides an experimental system in which effects of photoinhibition induced by lesions at the donor side of Photosystem II can be studied in vivo.Abbreviations Chl chlorophyll - DCIP 2,6-dichlorophenolindophenol - DPC 2,2-diphenylcarbonic dihydrazide - HEPES 4-(2-hydroxyethyl)-1-piperazinethanesulfonic acid - P₆₈₀ the primary electron donor to PS II - PpBQ phenyl-p-benzoquinone - PS II Photosystem II - Q_A the first quinone acceptor of PS II - Q_B the second quinone acceptor of PS II - SDS sodium dodecyl sulfate - Tris tris(hydroxymethyl)aminomethane - Tyr_D accessory electron donor on the D₂-protein - Tyr_Z tyrosine residue, acting as electron carrier between P₆₈₀ and the water oxidizing system     

Immunoaffinity co-purification of cytokinins and analysis by high-performance liquid chromatography with ultraviolet-spectrum detection

A rapid methodology for the simultaneous analysis of a large number of cytokinins is presented. The cross-reactivity of a mixture of polyclonal antibodies against zeatin riboside and isopentenyladenosine was exploited in a protocol that can be used for immunoaffinity purification of 23 additional cytokinins. Ligands include the cytokinin bases zeatin, dihydrozeatin, isopentenyladenine, benzyl-adenine and kinetin, and their corresponding nucleoside, nucleoside-5′-monophosphate, and 9-glucoside derivatives, as well as cis-zeatin, cis-zeatin riboside, the 2-methylthiol derivatives of isopentenyladenosine and zeatin riboside, and benzyl-adenine-3-glucoside. Mixtures of cytokinins could be retained with high recoveries of all the components. Immunoaffinity purification of extracts of Arabidopsis thaliana (L.) Heynh. and Solarium tuberosum L. gave fractions clean enough, as verified by gas chromatographymass spectrometry (GC-MS), to allow analysis of endogenous cytokinins using a single high-performance liquid chromatography (HPLC) step with on-line UV-spectrum detection. The detection limit was 4–6 pmol. The procedure described forms a routine assaying technique that is faster and simpler, yet yields better qualitative and quantitative information than the commonly used procedure of immunoassaying of HPLC fractions.     

The reductive tricarboxylic acid cycle of carbon dioxide assimilation: initial studies and purification of ATP-citrate lyase from the green sulfur bacterium Chlorobium tepidum.

Carbon dioxide is fixed largely by the reductive tricarboxylic acid (RTCA) cycle in green sulfur bacteria. One of the key enzymes, ATP-citrate lyase, was purified to apparent homogeneity from the moderately thermophilic green sulfur bacterium Chlorobium tepidum. The molecular weight of the native enzyme was about 550,000, and the preponderance of evidence indicated that the protein is composed of identical subunits (Mr of approximately 135,000) which degraded to two major proteins with Mrs of approximately 65,000 and approximately 42,000. Western immunoblots and in vitro phosphorylation experiments indicated that these two species could have been the result of proteolysis by an endogenous protease, similar to what has been observed with mammalian, yeast, and mold ATP-citrate lyase. In addition to apparent structural similarities, the catalytic properties of C. tepidum ATP-citrate lyase showed marked similarities to the eukaryotic enzyme, with significant differences from other prokaryotic ATP-citrate lyases, including the enzyme from the closely related organism Chlorobium limicola. Phosphorylation of C. tepidum ATP-citrate lyase occurred, presumably on a histidine residue at the active site, similar to the case for the mammalian enzyme. In contrast to the situation observed for other prokaryotic ATP-citrate lyase enzymes, the C. tepidum enzyme was not able to replace ATP and GTP for activity or use Cu2+ to replace Mg2+ for enzyme activity. Given the apparent structural and catalytic similarities of the enzyme from C. tepidum and its eukaryotic counterpart, the C. tepidum system should serve as an excellent model for studies of the enzymology and regulation of this protein.     

A 33-yr follow-up of peak oxygen uptake and related variables of former physical education students

Åstrand, Per-Olof, Ulf Bergh, and ÅsaKilbom. A 33-yr follow-up of peak oxygen uptake and relatedvariables of former physical education students. J. Appl. Physiol. 82(6): 1844-1852, 1997.In 1949, 27 female and 26 male physical education students were studied at amean age of 22 and 25 yr, respectively. They were restudied in 1970 and1982. Measurements included oxygen uptake, heart rate, and pulmonaryventilation during submaximal and maximal exercise on a cycle ergometerand treadmill. After 21 yr, peak aerobic power was significantlyreduced, from 2.90 to 2.18 l/min and from 4.09 to 3.28 l/min for womenand men, respectively. After another 12 yr, the 1970 maxima were notreduced further. From 1949 to 1982 there was a decrease in peak heartrate from 196 to 177 beats/min in women and from 190 to 175 beats/minin men (P < 0.05). Highest pulmonaryventilation did not change significantly. At an oxygen uptake of 1.5 l/min, the heart rate was the same in 1949 as in 1982. In conclusion,the physical fitness level of the subjects was well above average forthese ages. From 1970 to 1982 there was no decline in the average peakaerobic power, a finding possibly related to increased habitualphysical activity.

     

Stimulation of the insulin secretory mechanism following barium accumulation in pancreatic β-cells

Electrothermal atomic absorption spectroscopy was employed for measuring barium in β-cell-rich pancreatic islets microdissected from ob/ob-mice. Both the uptake and efflux of barium displayed two distinct phases. There was a 4-fold accumulation of barium into intracellular stores when its extracellular concentration was 0.26 mM. Unlike divalent cations with more extensive intracellular accumulation, the washout of Ba²⁺ was not inhibited by d-glucose. Ba²⁺ served as a substitute for Ca²⁺ both in maintaining the glucose metabolism after removal of extracellular Ca²⁺ and making it possible for glucose to stimulate insulin release. Furthermore, Ba²⁺ elicited insulin release in the absence of glucose and other secretagogues. The latter effect was reversible and was markedly potentiated under conditions known to increase the β-cell content of cyclic AMP. It is likely that the observed actions of Ba²⁺ are mediated by Ca²⁺, since Ca²⁺-dependent regulatory proteins, such as calmodulin, apparently cannot bind Ba²⁺ specifically.     

Vitamin analysis with use of a yeast electrode

A vitamin B₁ (thiamin)-sensitive electrode has been devised by combining an oxygen electrode with a yeast-containing membrane. The assembly was used for assaying thiamin at concentrations down to 10^?11 gl^?1. The analytical procedure developed should allow the measurement of 10–20 samples per hour. The performance of the yeast electrode was improved when alginate membranes reinforced with a nylon network were used. An apparatus for preparing such membranes is described together with a magnetic membrane holder facilitating handling of membranes in combination with electrodes.     

Intrafusal myosin heavy chain expression of human masseter and biceps muscles at young age shows fundamental similarities but also marked differences

Muscle spindles are skeletal muscle mechanoreceptors that provide proprioceptive information to the central nervous system. The human adult masseter muscle has greater number, larger and more complex muscle spindles than the adult biceps. For a better knowledge of muscle diversity and physiological properties, this study examined the myosin heavy chain (MyHC) expression of muscle spindle intrafusal fibres in the human young masseter and young biceps muscles by using a panel of monoclonal antibodies (mAbs) against different MyHC isoforms. Eight MyHC isoforms were detected in both muscles-slow-tonic, I, IIa, IIx, foetal, embryonic, α-cardiac and an isoform not previously reported in intrafusal fibres, termed IIx′. Individual fibres co-expressed 2–6 isoforms. MyHC-slow tonic separated bag₁, AS-bag₁ and bag₂ fibres from chain fibres. Typically, bag fibres also expressed MyHC-I and α-cardiac, whereas chain fibres expressed IIa and foetal. In the young masseter 98 % of bag₁ showed MyHC-α cardiac versus 30 % in the young biceps, 35 % of bag₂ showed MyHC-IIx′ versus none in biceps, 17 % of the chain fibres showed MyHC-I versus 61 % in the biceps. In conclusion, the result showed fundamental similarities in intrafusal MyHC expression between young masseter and biceps, but also marked differences implying muscle-specific proprioceptive control, probably related to diverse evolutionary and developmental origins. Finding of similarities in MyHC expression between young and adult masseter and biceps muscle spindles, respectively, in accordance with previously reported similarities in mATPase fibre type composition suggest early maturation of muscle spindles, preceding extrafusal fibres in growth and maturation.