Transport of free polymannose-type oligosaccharides from the endoplasmic reticulum into the cytosol is inhibited by mannosides and requires a thapsigargin-sensitive calcium store

The transport of free polymannose-type oligosaccharides from the lumen of the endoplasmic reticulum into the cytosol has been recently demonstrated (Moore,S.E.H., et al., 1995, EMBO J., 14, 6034-6042), but at present little is known of the characteristics of this process. Here, it is shown that inhibition of the transport of endogenously synthesized metabolically radiolabeled free oligosaccharides out of the endoplasmic reticulum into the cytosol of permeabilized HepG2 cells occurs when assays are conducted in the presence of mannose (IC50, 4.9 mM), or its derivatives modified at the first carbon (C1) of the sugar ring; alpha-methyl mannoside (IC50, 2.0 mM), mannoheptulose (IC50, 1.6 mM), and alpha-benzyl mannoside (IC50, 0.8 mM), whereas other monosaccharides (50 mM), differing from mannose at position; C2 (glucose), C3 (altrose), C4 (talose), C5 (l-rhamnose), and C6 (mannoheptose), have little effect. N-Acetylglucosamine does not inhibit oligosaccharide transport and, furthermore, although mannobioses and a mannotriose inhibit free oligosaccharide transport, di-N-acetylchitobiose is without effect. It is also shown that if the transport assay buffer is either depleted of calcium ions, or supplemented with the Ca2+/Mg2+ATPase inhibitor, thapsigargin, or with calcium ionophores, free oligosaccharide transport out of the endoplasmic reticulum is inhibited. These results demonstrate that the terminal nonreducing mannosyl residues of free polymannose-type oligosaccharides and not their N-acetylglucosamine-containing reducing termini, play an important role in the interaction of the free oligosaccharide with the transport machinery, and that this transport process requires the presence of calcium sequestered in the lumen of the endoplasmic reticulum.      

Development of Trypanosoma (M.) theileri in Tabanids

Thus far the life cycle of Trypanosoma (Megatrypanum) theileri has not been studied. We collected tabanids during the mass hatching, when only few tabanids are infected with trypanosomes. Tabanids were caught immediately after attacking a bait cow to serve as controls or after they had been allowed to engorge on the Trypanosoma (M.) theileri-infected cow. Tabanids were kept in the laboratory and used to study the developmental cycle of T. (M.) theileri in the tabanid gut. From day 1 to day 10 the presumably unfed controls and the engorged tabanids were dissected and cytological smears made from the mid- and hindgut. In total 2.6% (1/38) of the controls and 39% (23/59) of the engorged tabanids were positive for trypanosomes in the 1991 season. From day 1 to day 4 after engorgement trypanosomes were found in the midgut. Epimastigotes with a length of 29 μm on day 1 after infection multiplied by inequal division to form smaller epimastigotes of 26 μm on day 3. On day 4 morphologically indistinguishable trypanosomes of 21 μm total length were found in both mid- and hindgut. From day 5 to day 10 trypanosomes were found only in the hindgut in which the transformation to metacyclics was demonstrated, i.e. epimastigotes transformed to amastigote stages of 5 μm in total length.     

Winter distribution and overwintering strategies of the Antarctic copepod species Calanoides acutus,Rhincalanus gigas and Calanus propinquus (Crustacea,Calanoida) in the Weddell Sea

During the Winter Weddell Gyre Study in September–October 1989, the horizontal and vertical distribution, stage composition and feeding condition of the three antarctic copepod species Calanoides acutus, Rhincalanus gigas and Calanus propinquus were studied. The data indicate that C. acutus and R. gigas have the bases of their distributional ranges (sensu Makarov et al. 1982) in the Antarctic Circumpolar Current (ACC) and in the Warm Deep Water (WDW) entering the Weddell Gyre (WG). C. propinquus lived mainly in the cold WG south of the ACC. C. acutus overwintered mainly in the WG as stage IV copepodites (C). The species mainly inhabited the layers below the T_max stratum and down to 2000 m, but C V and females occurred slightly higher than C III and IV. Males prevailed over females and were confined to a rather narrow layer between 500 and 1000 m. Feeding experiments suggested all deep-living stages to be resting. However, if this species spawns in late autumn the younger C I–II can stay in the Winter Water (WW). R. gigas inhabited mainly the T_max stratum. In the eastern part of the WG, R. gigas breed in the WDW in autumn and hibernate as C I–III and C V–VI in the first and second winter, respectively. In the ACC zone, however, its life cycle is different and winter breeding of overwintered adults occurs. Most of the C. propinquus population overwintered in the WG as C III–V, inhabiting the WW. In the upper water layers in the interior of the WG, C III dominated with upto 18,000 individuals 1,000 m³. Shallow living C. propinquus were in the active, feeding state. Persistence of active feeding zooplankton populations in the WW of the WG can be an important factor influencing processes of phytoplankton development and the particle flux.     

Microtubules and cyclic amp in human leukocytes: on the order of things

We have shown previously that the β-adrenergic agonist isoproterenol (2μM) and the phosphodiesterase inhibitor isobutylmethylxanthine (1 mM) produce a much greater increase in cyclic AMP in human leukocytes that have been pretreated with colchicine (or with other agents that affect microtubule assembly) than in control leukocytes. The effects of colchicines were both time- and dose-dependant. These and other data suggested that the generation of cyclic AMP is normally restricted by an intact system of cytoplasmic microtubules. If so, then the same time and dose dependencies might apply to other colchicines-induced changes in leukocyte function. We have now assayed the distribution of concanavalin A (Con A)-receptor complexes on the leukocyte membrane, taking into account that leukocytes competent to assemble microtubules show a uniform distribution of surface- bound Con A whereas microtubule-deficient cells accumulate Con A in surface caps. We have found that the effect of colchicine on capping is also both time- and dose dependent, and that the dose-response relationships conform to those required to increase cyclic AMP levels. These findings provide further evidence that both colchicine-induced Con-A capping and colchicine- induced cyclic AMP generation depend upon the relaxation of constraints normally imposed by cytoplasmic microtubules upon the plasma membrane, which limit, respectively, lateral mobility of the lectin-receptor complexes, and expression of hormone-sensitive adenylate cyclase. Moreover, colchicine-induced Con-A cap formation is not affected even by very large changes in leukocyte cyclic AMP levels. Thus, elevated cyclic AMP levels do not appear to promote the dissolution of microtubules; rather, the dissolution of microtubules permits the generation of increased amounts of cyclic AMP.     

A direct photo-activated affinity modification of tetracycline transcription repressor protein TetR(D) with tetracycline(1)

Results of a first successful application of a direct photo-induced affinity modification of Tet repressor (TetR(D)) protein with tetracycline within a complex of known three-dimensional structure are described. The conditions of the modification have provided suitable yields of the modified complex and allowed characterization of the modified segments of the protein. The potential of tetracycline as a fine modifying reagent was established. In the complex of TetR(D) protein with tetracycline, the antibiotic modifies at least two segments, Ile59-Glu73 and Ala173-Glu183, which form a binding tunnel for the drug according to the X-ray analysis. These data open possibilities for the use of different tetracycline targets for structural studies in solution.     

Analysis of the contribution of forest pathways to the radiation exposure of different population groups in the Bryansk region of Russia

The experience gained in the aftermath of serious radiation accidents shows that forests are an important source of external and internal exposure of the affected population. This paper presents the results of an assessment of the major radiological consequences for forests of Russia, most heavily contaminated after the Chernobyl nuclear power plant (ChNPP) accident. Illustrated in the Novozybkovsky district of the Russian Federation, the significance of different forest exposure pathways is estimated and the doses resulting from forest pathways are compared with the doses from agricultural products. It has been found that the contribution of mushrooms and berries to the internal doses of the population, relative to the doses from agricultural products, varied from 10–15% in 1987 to 40–45% in 1996. The results indicate large differences in internal exposure of members of the ”critical groups” and ”normal population”, increasing with time after deposition. Data are presented that give information on the contribution of forests to the collective doses of inhabitants of the area under consideration. It has been shown that for 10 years after the accident (1987–1996), the contribution of forest products to the collective dose of the rural population living in contaminated forests of the Novozybkovsky district, amounts to about 20% (213 person Sv) of the total collective dose of internal and external exposures. However, a potential impact of these products including the dose from exported products is much higher and might reach 659 person Sv. It has been found that in the long-term after the ChNPP accident, serious attention should be given to forest countermeasures, and restoration strategies should be selected on the basis of a combined analysis of the effectiveness of forest and agricultural countermeasures. Received: 16 March 2000 / Accepted: 10 September 2000     

Analysis of factors determining the biological availability of 137Cs in forest ecosystem soils

On five forest plots varying in species composition of woody plants and soil properties, 137Cs accumulation by vegetation of the forest understorey has been studied. Differences in 137Cs availability for root uptake are shown to be dependent on a vertical distribution of 137Cs content over the soil profile, percentage of exchangeable 137Cs and distribution of plant root systems (fungous mycelium) in soil. A 137Cs-bioavailability index is suggested and its non-linear relationship with 137Cs transfer factors to different plant species in the understorey is shown.     

Cloning and Biochemical Characterization of BglC, a β-Glucosidase from the Cellulolytic Actinomycete Thermobifida fusca

An operon, bglABC, that encodes two sugar permeases and a β-glucosidase was cloned from a cellulolytic actinomycete, Thermobifida fusca, into Escherichia coli and sequenced. The bglC gene encoding an intracellular β-glucosidase (β-d-glucoside glucohydrolase, EC 3.2.1.21) belonging to glycosyl hydrolase family 1 was subcloned and expressed in E. coli. The purified enzyme (MW 53,407 Da; pI 4.69) hydrolyzed substrates containing both β 1 → 4 and β 1 → 2 glycosidic bonds, and was most active against cellobiose (V_max= 29, K _m = 0.34 mm), cellotriose, cellotetraose, and sophorose. The enzyme also showed aryl-β-glucosidase activity on p-nitrophenyl-β-d-glucopyranoside and p-nitrophenyl-β-d-cellobioside. BglC had a pH optimum of 7.0 and a temperature optimum of 50°C. The enzyme was stable at 60°C, but was rapidly inactivated at 65°C. BglC was inhibited by low concentrations of gluconolactone, but was insensitive to end-product inhibition by glucose and was not affected by Ca or Mg ions or EDTA. Its properties are well suited for use in a process to hydrolyze biomass cellulose to glucose. Received: 21 August 2000 / Accepted: 4 October 2000