Structural identification of oligosaccharides produced by Zymomonas mobilis levansucrase

Summary The chemical structure of oligosaccharides produced by Zymomonas mobilis levansucrase (EC 2.4.1.10) was determined using enzymatic hydrolysis, mass spectroscopy, and ¹³C-NMR spectroscopy. The major oligosaccharide (98% of total oligomers) produced from transfructosylation reactions with -sucrose was identified as 1-kestose (O--D-glucopyranosyl-(12)-O--D-fructofuranosyl-(12)--D-fructofuranoside).     

Cellular organization of the embryonic lobster heart

Summary The cellular organization of the embryonic heart of the lobster Homarus americanus was examined in 6-week and 6-month-old animals. The heart wall consists of an outer adventitial layer of fibroblast cells and an inner layer of transversely striated myocardial cells. Present in close association with the myocardium are cardiac neurons, hemocytes and so-called storage cells.Adjacent fibroblasts form fasciae adhaerentes and gap junctions. Adherent junctions also occur between fibroblasts and myocardial cells. Intercalated discs and differentiated membrane regions of close apposition (4 nm) occur between adjacent myocardial cells.The cardiac neurons form a ganglion that contains four small and five large somata. Regions of neuropil are present. Motor axons arising from the cardiac ganglion form neuromuscular synapses with the myocardial cells.The storage cells contain large inclusions and form gap junctions with the myocardial cells. They may supply nutritive material to the developing myocardium.The heart at 6 weeks is about 200 m long and 160 m wide. At 6 months, it is about 300 m long and 250 m wide. The myocardium at 6 weeks is one cell layer thick, and the cells are from 2–6 m in maximum width. At 6 months the myocardium is 2–4 cells thick, and the cells are from 6–12 m in width. Therefore, the myocardium grows by an increase in the number and size of the myocardial cells.     

Changes in LHβ-gene and FSHβ-gene expression in the ram pars tuberalis according to season and castration

Luteinizing hormone beta (LH) and follicle stimulating hormone beta (FSH) subunits and their mRNAs were studied in the ram pars tuberalis following different seasonal (winter vs summer) and experimental (intact vs castrated animals) conditions. Hormone-containing cells were identified by immunohistochemistry, and mRNAs for LH and FSH by in situ hybridization using homologous double-stranded ³⁵S-cDNAs. The labelling was quantified by image analysis. Immunohistochemical staining showed that cells containing LH and FSH were localized mainly in the ventral part of the pars tuberalis but that, in the summer, additional LH-containing cells were present in the dorsal part in intact rams. On the other hand, LH-mRNA labelling was found in the whole pars tuberalis in wethers but only in the ventral part in intact rams. The magnitude of LH-mRNA labelling was significantly greater in summer than in winter rams, and in castrated than in intact animals (P<0.001). However, the number of labelled cells was found to be the greatest in the winter (P<0.001) and was not affected by castration. FSH-mRNA expression was similar to that of LH-mRNA except that the level and extent were considerably lower. Thus, our results show an increase in the magnitude of gonadotropin subunit-mRNA in the summer and following castration; this increase appears to involve the entire pars tuberalis.     

Regulation of potato meristem development by jasmonic acid in vitro

The influence of jasmonic acid (JA) on differentiation of meristems of the potato,Solanum tuberosum cv. Vesna, was investigated in vitro. Meristems were grown on Murashige and Skoog (MS) medium supplemented with indole-3-acetic acid (IAA) (10 M), kinetin (10 M), gibberellic acid (3 M), as modified by Bang. Addition of JA in concentrations of 0.5–10 M increased the number of meristems that developed into buds, particularly in meristems isolated from shoots grown from tubers in the dark. JA had no noticeable effect on meristems from germs grown in light. All added concentrations of JA retarded callus and root formation. The inhibitory effect on rhizogenesis disappeared immediately after transfer of the developed buds to medium without JA.     

Chimeric evolution of the 2-μm genome in Saccharomyces cerevisiae

We compared the nucleotide substitution pattern over the entire genome of two unique variants of the 6,300-bp selfish DNA (2 m) plasmid in Saccharomyces cerevisiae. The DNA sequence of the left-unique region is identical among 2-m variants, while the right-unique region shows substantial divergence. This chimeric pattern cannot be explained by neutral or Darwinian selection models. We propose that horizontal transmission of the 2-m plasmid coupled with a directed, polarized gene conversion maintains the DNA sequence of the left-unique region, whereas the right-unique region is subject to random drift and Darwinian selection. Correspondence to: G.H. Rank     

High-frequency somatic embryogenesis from small suspension-cultured clusters of cells of an interspecific hybrid of Oryza

Suspension cultures in which cell clusters were small and had a high capacity for somatic embryogenesis (about 60%) were established from immature panicles of F₁ plants from a cross between Oryza sativa and Oryza latifolia The cells were subcultured at seven-day intervals in a modified N6 medium. The cell clusters were quite small (approximately 30–200m in diameter) after culture for two months in this medium. When small clusters of cells were transferred to N6 medium, that had been diluted with an equal volume of water and supplemented with -naphthalenacetic acid (53 nM), 4-pyridylurea (2.2 M) and sucrose (30 gl^-1), somatic embryogenesis occurred at high frequency (about 60%). The system established in the present work is useful for biochemical and molecular biological research of the somatic embryogenesis in the Gramineae.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - NAA -naphthalenacetic acid - 4-PU 4-pyridylurea - MS Murashige and Skoog (1962)     

Auxin increases the hydraulic conductivity of auxin-sensitive hypocotyl tissue

The ability of water to enter the cells of growing hypocotyl tissue was determined in etiolated soybean (Glycine max (L.) Merr.) seedlings. Water uptake was restricted to that for cell enlargement, and the seedlings were kept intact insofar as possible. Tissue water potentials ( _w) were measured at thermodynamic equilibrium with an isopiestic thermocouple psychrometer. _wwas below the water potential of the environment by as much as 3.1 bars when the tissue was enlarging rapidly. However, _w was similar to the water potential of the environment when cell enlargement was not occurring. The low _w in enlarging tissue indicates that there was a low conductivity for water entering the cells.The ability of water to enter the enlarging cells was defined as the apparent hydraulic conductivity of the tissue (Lp). Despite the low Lp of growing cells, Lp decreased further as cell enlargement decreased when intact hypocotyl tissue was deprived of endogenous auxin (indole-3-acetic acid) by removal of the hypocotyl hook. Cell enlargement resumed and Lp increased when auxin was resupplied exogenously. The auxin-induced increase in Lp was correlated with the magnitude of the growth enhancement caused by auxin, and it was observed during the earliest phase of the growth response to auxin. The increase in Lp appeared to be caused by an increase in the hydraulic conductivity of the cell protoplasm, since other factors contributing to Lp remained constant. The rapidity of the response is consistent with a cellular site of action at the plasmalemma, although other sites are not precluded.Because the experiments involved only short times, auxin-induced changes in cell enlargement could not be attributed to changes in cell osmotic potentials. Neither could they be attributed to changes in turgor, which increased when the rate of enlargement decreased. Rather, auxin appeared to act by altering the extensibility of the cell walls and by simultaneously altering the ability of water to enter the growing cells under a given water potential gradient. The hydraulic conductivity and extensibility of the cell walls appeared to contribute about equally to the control of the growth rate of the hypocotyls.     

Intersectional cytoplasmic hybrids in Nicotiana

Summary Cybrid plants having the nuclear genomes of one species and either or both plastomes and chondriomes of another species were obtained by fusing protoplasts of Nicotiana sylvestris, as recipients, with X-irradiated protoplasts of N. rustica as donors of chloroplasts and mitochondria. Forty-nine flowering plants, derived from 28 calli, were analysed. As expected, they all had N. sylvestris (i.e. recipients) morphology. Chloroplast DNA restriction patterns indicated that 8 and 41 plants had N. rustica and N. sylvestris plastomes, respectively. Some of the plants with either type of plastomes produced sterile pollen but none showed anther malformation typical to alloplasmic male sterility. Chondriome identification by mitochondrial DNA restriction analysis of cybrid plants revealed only restriction patterns which were either similar or identical to those of N. sylvestris while no cybrids with N. rustica restriction patterns were detected.     

Sequestered actin in chick embryo fibroblasts

Chick embryo fibroblasts contain about 75-100 M unpolymerized actin and at least four proteins which can bind actin monomers, actin depolymerizing factor (ADF), gelsolin, profilin, and thymosin ₄ (T₄). Fibroblast extracts are analyzed by non-denaturing polyacrylamide gel electrophoresis and immunoblotting where most of the G-actin is detected as a complex with T₄. When fibroblast extracts are fractionated by gel filtration and the fractions are analyzed by PAGE and HPLC, most of the G-actin elutes in a peak that also contains T₄ at an overall molar ratio of 1.9:1 relative to actin. Gelsolin, profilin, and ADF are also detectable in the gel filtration eluate and at least partly coelute with actin, and account for only a minor fraction of the soluble actin pool. These observations indicate that under the growth conditions studied, T₄ is the major actin-sequestering protein in fibroblasts.     

Interactions of carbaryl with estuarine bacterial communities

The addition of carbaryl (100g/ml) to a model estuarine ecosystem did not affect the number of bacteria in the sediment, but reduced the diversity (as measured by the rarefaction technique) of the microbial community as compared with a control model ecosystem. Two carbaryltolerant strains of bacteria were isolated from the carbaryl-treated system, but none were isolated from the control system. Bacterial growth and filter paper decomposition in mixed cultures was prevented by 100g/ml carbaryl, but this amount had no effect on the extracellular cellulase of an estuarine isolate. Increasing the amount of organic matter in the medium attenuated the toxicity of carbaryl to pure cultures of an estuarine isolate. The addition of 1, 10, or 100g/ml carbaryl to field plots had no effect on bacterial numbers, diversity, or filter paper decomposition. The amount of carbaryl in sediments exposed to 100g/ml fell below the limit of detection by thin-layer chromatography within 12 hours. In sterile and nonsterile model systems, carbaryl rapidly adsorbed to sediment, and hydrolyzed to 1-naphthol in both sediment and water. Although carbaryl may be toxic to bacteria under some conditions, the amounts that might enter and persist in an estuary are insufficient to have a significant impact on the sediment microbial community.     

Increasing infiltration and activation of CD8+ tumor-infiltrating lymphocytes after eliminating immune suppressive granulocyte/macrophage progenitor cells with low doses of interferon γ plus tumor necrosis factor α

By secreting granulocyte/macrophage colonystimulating factor (GM-CSF), metastatic Lewis lung carcinoma (LLC-LN7) tumors induce the appearance of myelopoiesis-associated immune-suppressor cells that resemble granulocytic-macrophage (GM) progenitor cells. The presence of these GM-suppressor cells in mice bearing LLC-LN7 tumors was associated with a reduced capacity of splenic T cells to proliferate in response to interleukin-2 (IL-2). Administration of low doses of 100 U interferon (IFN) plus 10 U tumor necrosis factor (TNF) to the tumor bearers, a combination treatment that we previously showed to diminish the presence of GM-suppressor cells synergistically, restored proliferative responsiveness of the splenic T cells to IL-2. These LLC-LN7-bearing mice were also examined for whether cells that phenotypically resemble GM-progenitor cells (ER-MP12⁺ cells) infiltrate the tumor mass. ER-MP12⁺ cells composed approximately 10% of the cells isolated from dissociated tumors of mice that had been treated with placebo or with either IFN or TNF alone, but IFN/TNF therapy markedly reduced the number of tumor-infiltrating ER-MP12⁺ suppressor cells. The IFN/TNF treatment to eliminate GM-suppressor cells and restore T cell responsiveness to IL-2 was next coupled with low dose IL-2 therapy (100 U twice daily). Addition of IL-2 to the treatment regimen did not significantly influence the effectiveness of the IFN/TNF treatment in eliminating GM-suppressor cells from the LLC-LN7 tumor mass. However, inclusion of IL-2 with the IFN/TNF treatment regimen enhanced the CD8⁺, but not the CD4⁺, cell content within the tumor, and diminished the number of metastatic lung nodules within the mice. When these tumors were excised, dissociated, and bulk-cultured with a low dose of IL-2, an increased level of cytotoxic T lymphocyte (CTL) activity was generated in the TIL cultures from mice that had received IFN/TNF plus IL-2 treatments. A lesser but detectable level of CTL activity was generated in TIL cultures from mice that were treated with only IFN/TNF, while no CTL activity was generated in tumor cultures from mice receiving only placebo or low-dose IL-2. These results suggest the effectiveness of IFN plus TNF therapy in restoring IL-2 responsiveness in mice bearing GM-suppressor cell-inducing tumors and at enhancing both the intratumoral CD8⁺ cell content and the generation of CTL activity in bulk cultures of these tumors.This study was supported by the Medical Research Service of the Department of Veterans Affairs, by grants CA-45080 and CA-48080 from the National Institutes of Health, and by the American Cancer Society, Illinois     

The Synthesis and Antiviral Activity of Glycyrrhizic Acid Conjugates with α-D-Glucosamine and Some Glycosylamines

Glycyrrhizic acid and its 30-methyl ester were conjugated with 2-amino-1,3,4,6-tetra-O-acetyl-2-deoxy--D-glucopyranose, 2,3,4,6-tetra-O-acetyl--D-glucopyranosyl amine, 2,3,4-tri-O-acetyl--L-arabinopyranosyl amine, 2-acetamido-2-deoxy--D-glucopyranosyl amine, and -D-galactopyranosyl amine using N,N-dicyclohexylcarbodiimide and its mixtures with N-hydroxybenzotriazole. Structures of the conjugates were confirmed by IR, UV, ¹H, and ¹³C NMR spectroscopy. The glycoconjugate with the residues of 2-acetamido-2-deoxy--D-glucopyranosyl amine in the carbohydrate part of its molecule exhibited antiviral activity (ID₅₀ 4 g/ml) toward the herpes simplex type 1 virus (HSV-1) in the VERO cell culture. Two compounds demonstrated anti-HIV-1 activity (50–70% inhibition of p24) in a culture of MT-4 cells at concentrations of 0.5–20 g/ml.     

Effects of biogenic amines and adrenergic drugs on oviposition in the cattle tickBoophilus: Evidence for octopaminergic innervation of the oviduct

Oviposition in the cattle tickBoophilus microplus was arrested by octopamine and the -adrenergic agonists clonidine, tolazoline, and naphazoline, at doses of 25g per tick. The same effect was obtained with a sublethal dose of Amitraz of 10 g per tick. In contrast, the -adrenergic agonists apomorphine, synephrine, phetolamine, metaclopromide and chlorpromazine were ineffective. No mortality was observed at any of the doses tested. Reserpine, which depletes endogenous adrenergic terminals, mimicked the effects of octopamine in blocking oviposition. Acetylcholine agonists also blocked oviposition but dopaminergic agonists did not. Evidence is presented suggesting that control of the oviduct is mediated by octopaminergic receptors.     

The electrical response ofAvena coleoptile cortex to auxins

Solute mobilities of 28 compounds in isolated cuticular membranes (CM) from Capsicum annuum L. fruit, Citrus aurantium L. and Pyrus communis L. leaves were studied using unilateral desorption from the outer surface. First-order rate constants of desorption (k^*), which are directly proportional to the diffusion coefficient in the waxy outer limiting skins of cuticles were measured. When log k^* was plotted vs. molar volumes of test compounds linear graphs were obtained. The y-intercepts of these graphs (k^*) represent the mobility of a hypothetical molecule having zero molar volume and the slopes of the graphs () represent the size selectivity of the barrier and are related to the free volume available for diffusion. Thus, solute mobilities in cuticles are composed of two independent terms which are subtractive. If k^* and are known, k^* can be estimated for any solute from its molar volume (V_x) using the equation log k^*=log k^* –V_x. These parameters were used to analyse the effects of plant species, extraction of cuticular waxes and molecular structure of solutes on solute mobilities in plant cuticles. For aliphatic solutes, k^* was a factor of 10 smaller than for cyclic compounds, while was 0.011 and 0.012, respectively. The k^*-values for CM of the three species were very similar, but was higher for bitter-orange CM (0.012) than for those of pepper fruits and pear leaves (0.009). This has the consequence that differences in solute mobilities (k^*) among cuticles from different plan species increase with increasing molar volumes of solutes. Our data and our analysis provide evidence that constituents of cuticular waxes are mobile, at least in the solid amorphous wax fraction, but mobility decreases rapidly with increasing molar volume. For instance, if amounts to 0.01, mobilities of wax monomers decrease by a factor of 10 for every increase in molar volume of 100 cm³ · mol^–1. Thus, hexadecanoic acid is quite mobile in the amorphous wax fraction of Citrus (k^*=1.5×10^–6·s^–1), but for dotriacontane having twice the molar volume, k^* was only 2.5×10^–9·s^–1, which is almost three orders of magnitude smaller. Wax esters have even higher molar volumes and their mobilities will be even smaller (about 4×10^–12·s^–1 for a C₄₈-ester). Since low chain mobilities are a prerequisite for low mobilities and permeabilities, the selective advantage of high-molecular-weight wax monomers in plant cuticular waxes becomes obvious. Extracting cuticular waxes from pear leaf CM increased solute mobilities by a factor of 182, but it had no effect on size selectivity. We interpret this result as evidence to the effect that cuticular waxes reduce mobility by increasing tortuosity of the diffusion path, rather than by decreasing the mean free path of diffusional jumps and jump frequencies of diffusants.Abbreviations CM cuticular membrane(s) - 2,4-D 2,4-dichloro-phenoxyacetic acid - LAB lactic acid buffer - MX polymer matrix membranes - UDOS unilateral desorption from the outer surface     

Troop size,home range area and seasonal range use of the Japanese macaque in the Northern Japan Alps

Home range area and habitat use of the wild Japanese macaque (Macaca fuscata) were investigated between elevations of 600m and 3000m in the Northern Japan Alps, in areas where there is great variation in vegetation type. A total of 22 troops were located either visually or by radiotracking from December 1996 to February 2000. The average troop size was 48.4 individuals, the average ranging area (65% probability distribution calculated using the adaptive kernel method) was 3.7km² and the average elevation of each troop varied from 740m to 1458m. Two troop types were distinguished according to their habitat use: (i) rural type groups frequently utilized cultivated land (n=12); and (ii) natural type groups never utilized cultivated land (n=10). Natural groups inhabited higher elevation areas and showed a greater dependency on deciduous broad-leaved forests during all seasons. In contrast, rural groups utilized the larch plantation more in spring, and the red pine forest in summer. Rural groups were observed on cultivated lands more often from summer to winter than in spring. The size of the rural groups was twice as large as that of the natural groups. Troop size and home range area showed significant correlation within each group type. The per capita home range area of rural groups was smaller than that of natural groups. These results suggest that per capita home range area is negatively correlated with habitat quality.     

Plant regeneration via somatic embryogenesis in Styrian pumpkin: cytological and biochemical investigations

Somatic embryo formation was induced from cotyledon explants of Styrian pumpkin (Cucurbita pepo L. subsp. pepo var. styriaca Greb.) by using a solid MS medium supplemented with 16.11M NAA and 4.44M BA or 26.85M NAA and 13.32M BA. The callus proliferation was more efficient on medium supplemented with 26.85M NAA and 13.32M BA. In contrast, the embryogenic response was higher on medium with lower concentrations of growth regulators (16.11M NAA and 4.44M BA). The time needed for embryo induction did not depend on medium composition. Embryos in globular stage were transferred to three different maturation media, containing 2.89M GA₃ in combination with 0.54M NAA, 11.42M IAA and growth regulator-free medium. The germination rate was the highest when embryos were cultured on medium with 11.42M IAA. Plantlets grown on this medium achieved maturity suitable for transplantation into soil within 9 to 10weeks. The regenerated plants were successfully transferred into field and developed fertile flowers and set fruits. Biochemical analysis showed significant lower total glutathione levels among in vitro grown plantlets compared to seedlings grown in soil. When the plantlets were transferred into soil, they reached a normal size within a month and the glutathione concentration was comparable to seed-derived plants at the same developmental stage. Transmission electron microscopy was used to investigate possible differences in the ultrastructure of cells from callus cultures, and leaf cells of regenerated and seed-derived plants. Differences in the ultrastructure were found within chloroplasts which contained only single thylakoids, large starch grains and small plastoglobuli in callus cells in comparison to leaf cells, which possessed a well developed thylakoid system, small starch grains and large plastoglobuli.     

Problems in estimating growth rates of marine phytoplankton from short-term14C assays

Growth rate estimates () of phytoplankton populations that were sampled from nitrogen-limited continuous cultures and then incubated for short durations in batch culture with added¹⁴C-HCO₃ ^– were significantly different than steady-state growth rates () for 3 of 5 marine phytoplankton species. Two diatoms,Thalassiosira weissflogii andChaetoceros simplex, displayed virtually identical growth rates (=) over a wide range of, whereas for a third diatom,Phaeodactylum tricornutum, was overestimated by an average of 40% compared to. In contrast, was underestimated by the¹⁴C technique for the two remaining species: up to 40% at a steady-state of 1.0 day^–1 for the chlorophyteDunaliella tertiolecta and up to 100% at of 1.4 day^–1 for the haptophytePavlova lutheri. For the latter two species the divergence between and appeared to increase with increasing steady-state. A simple model of labeled and total carbon flow between the aqueous phase and cellular biomass was constructed to demonstrate that respiration was negligible when=, but was significant when>. In the cases in which<, a rapid physiological alteration presumably took place once the steady state was disturbed and cells were placed in the incubation chambers, which perhaps was related to the nutritional state of the cultures at the time of sampling. Questions thus are raised regarding our ability to measure accurately primary productivity from shipboard experiments with confined samples of phytoplankton from nutrient-impoverished waters that probably are less hardy than the laboratory cultures used in these studies.     

New Triterpene Glycosides from an Ulosa sp. Sponge

New triterpene glycosides, ulososides C, (20S,22S,23R,24S)-3,22,23-trihydroxy-3-O-(-D-glucopyranosyl)-32-nor-24-methyllanost-8(9)-ene-30-oic acid, D, (20S,22S,23R,24S)-3,22,23-trihydroxy-3-O-(-D-N-acetylglucosaminopyranosyl)-32-nor-24-methyllanost-8(9)-ene-30-oic acid, and E, (20S,22S,23R,24S)-3,22,23-trihydroxy-3-O-(-D-glucuronopyranosyl-(1 2)--D-arabinopyranosyl-32-nor-24-methyllanost-8(9)-ene-30-oic acid, were isolated from an Ulosa sp. sponge. Their structures were determined by spectral methods and chemical transformations. Specific features of their structures are discussed.     

5′-AMP hydrolysis by suspensions and homogenates of pancreatic islet cells from normal and cortisone-treated rats

Summary Suspensions of endocrine pancreas cells were prepared by shaking collagenase-isolated rat islets of Langerhans in calcium-free buffer. When incubated with 1.0 mM substrate at pH 7.4, the cells split,P _i from 5-AMP at a rate of 87 nmol/h per g DNA, and from-glycerophosphate at a rate of 25 nmol/h per g DNAK _m for 5 AMP was about 54 M. Adenosine or theophylline inhibited the 5-AMP hydrolysis. Homogenization of the cells increased the activity toward 5-AMP by 23% and that toward-glycerophosphate by 115%. Injecting rats with cortisone had no effect on the 5-AMP hydrolysis by whole cells but significantly increased the activity in cell homogenates; the intracellular activity toward 5-AMP was more than doubled by the cortisone treatment. Staining whole islet cells for 5-AMP-splitting activity resulted in a demarcation of the cell periphery in control rats. Cells from cortisone-treated rats showed heavier deposits of reaction product, and their cell periphery did not stand out as clearly. It is suggested that 5-nucleotidase is largely an ectoenzyme in normal rat islet cells. The cells also contain an as yet unidentified intracellular phosphatase that seems to be solely responsible for the increased hydrolysis of 5-AMP in cortisone-treated rats.     

Methylobacterium rhodesianum MB 126 possesses two acetoacetyl-CoA reductases

Two constitutive acetoacetyl-CoA (AcAc-CoA) reductases were purified from Methylobacterium rhodesianum MB 126, an NADPH-linked d(-)--hydroxybutyryl-CoA forming reductase (enzyme A) and an NADH-and NADPH-linked l(+)--hydroxybutyryl-CoA forming reductase (enzyme B). Enzyme A and B give apparent K _m values of 15 M and 30 M for AcAc-CoA, 18 M for NADPH and 30 M for NADH, respectively. They are inhibited by AcAc-CoA at concentrations higher than 25 M and 50 M, respectively. The contribution of the two reductases to poly--hydroxybutyrate synthesis is discussed.