Structural and immunochemical studies on the lipopolysaccharide of the ‘T-antigen’-containing mutant Proteus mirabilis R14/1959

Abstract In DOC-PAGE, lipopolysaccharide (LPS) of Proteus mirabilis R14/1959 (Rb-type) mutant showed a ladder-like migration pattern indicating the presence of a high molecular weight polysaccharide chain. The isolated polysaccharide, called T-antigen because of similarity with the T1 chain of Salmonella friedenau LPS, contained d -glucose, d -galacturonic acid ( d -GalA), and d -GlcNAc in molar ratios 2:1:1 and was structurally different from the O-antigen of the parental S-strain P. mirabilis S1959 but identical to the O-antigen of another S-strain Proteus penneri 42. The importance of a d -GalA( l -Lys)-containing epitope, most likely present in the core region of LPS, and of GalA present in the T-antigen chain in manifesting the serological specificity of P. mirabilis R14/1959 were revealed using rabbit polyclonal homologous and heterologous R- and O-specific antisera and the appropriate antigens, including synthetic antigens which represent partial structures of various Proteus LPS.     

淤泥湖退化水生植被恢复及其在湖泊资源天然生态库中作用的研究

对淤泥湖水生植被的天然恢复过程、群落组成、结构、功能与动态等进行了定位研究（１９９２～１９９５）．该湖水生植物共有２０科２９属４１种．４种生活型的植物种类在该湖均有分布,以沉水草本（１５种）和挺水草本（１３种）为多．８个分布区类型中世界广布成分１６种和东亚成分１０种．群落结构包括层次结构和层片结构．该彻共有８个群落类型;群落现存量３２０８．８ｇ·ｍ－２,全湖植被资源贮量为１６３６５．２ｔ．     

Carbohydrate Uptake and Utilization byClostridium beijerinckiiNCIMB 8052

The clostridia are a diverse group of obligately anaerobic bacteria with potential for the fermentative production of fuels, solvents and other chemicals. Several species exhibit a broad substrate range, but there have been few studies of the mechanisms involved in regulation of uptake and metabolism of fermentable carbohydrates.Clostridium beijerinckii(formerlyClostridium acetobutylicum) NCIMB 8052 exhibited transport activity for hexoses and hexitols. Glucose-grown cells transported glucose and fructose, but not galactose, glucitol (sorbitol) or mannitol, transport of which was induced by growth on the respective substrates. Phosphorylation of glucose, fructose, glucitol and mannitol by cell extracts was supported by phosphoenolpyruvate, indicating the involvement of a phosphotransferase system in uptake of these substrates. Fructose phosphorylation was also demonstrated by isolated membranes in the presence of fructose 1-phosphate, thus identifying this derivative as the product of the fructose phosphotransferase system. The presence of phosphotransferase activities in extracts prepared from cells grown on different carbon sources correlated with transport activities in whole cells, and the pattern of transport activities reflected the substrate preference of cells growing in the presence of glucose and another carbon source. Thus, glucose and fructose were co-metabolised, while utilization of glucitol was prevented by glucose, even in cells which were previously induced for glucitol metabolism. Of the substrates examined, only galactose appeared to be transported by a non-phosphotransferase mechanism, since a significant rate of phosphorylation of this sugar was supported by ATP rather than phosphoenolpyruvate.     

Importance of dormancy and sink strength in sprouting of onions (Allium cepa) during storage

In onion ( Allium cepa L.) postponement of sprouting is necessary to achieve long term storage. We studied the factors determining sprouting during dry storage at 16°C. The period to visible sprouting depends on the length of the dormancy period, if present, and on the growth rate of the sprout. In the three cultivars tested, sprouts were initiated within 2 weeks after harvest indicating the absence of a real dormancy period. Sprout length increased linearly during storage. The mitotic activity of the apex decreased before harvest, was low at the transition from scale to leaf formation, and increased again when the sprout was initiated. From a few weeks before harvest, the initially high fructan content of the scales decreased, leading to a large increase in fructose. The sprout always contained enough carbohydrates for growth (between 50 and 60 mg g⁻¹ dry weight, of which 30% was fructan). The activity of sucrose synthase (EC 2.4.1.13) increased as the sprout grew, indicating an increase in sink strength. Invertase (EC 3.2.1.26) was absent in all bulb organs, during the various developmental stages. Although carbohydrates and enzymes were available for fast sprouting, sprout growth was still linear instead of exponential during dry storage at temperatures favorable for growth (16°C). The relative importance of factors determining sprouting are discussed.     

Comparison of carbohydrate utilization and energy charge in the yellow flag iris (Iris pseudacorus) and garden iris (Iris germanica) under anoxia

Carbohydrate and energy metabolism of the flooding- and anoxia-tolerant Iris pseudacorus and the intolerant Iris germanica rhizomes were investigated under experimental anoxic conditions. Rhizomes of I. pseudacorus and I. Germanica were incubated in the absence of oxygen from 0 to 60 and 16 days, respectively. Amounts of glucose, total reducing sugars and non-reducing sugars (starch, fructan and oligosaccharides) in the rhizomes were measured. Ethanol concentration and adenylate energy charge were determined enzymatically. Glucose content of I. pseudacorus rhizomes decreased gradually during the first 30 days under anoxia and then increased at the same time as adenylate energy charge values started to decline. In I. germanica rhizomes the changes were more dramatic and the time scale was much shorter than in I. pseudacorus but the changes were similar. Non-reducing sugar content of I. pseudacorus rhizomes decreased rapidly during the first 15 days under oxygen deprivation and then increased again, to near starting levels at 35 days. In I. germanica the amount of non-reducing sugars decreased gradually during the anoxic incubation. Under aerobic control conditions, adenylate energy charge (AEC) of I. pseudacorus and I. germanica rhizome tissue was 0.87±0.01 and 0.81±0.01, respectively. In I. pseudacorus AEC remained high until 30 days under anoxia. In contrast, the energy charge of I. germanica rhizome tissue remained above 0.6 for 4 days only. Large amounts of ethanol were found in anoxic rhizome tissues of I. pseudacorus (up to 0.21 M ) and I. germanica (0.06 M ) after 45 days and 8 days, respectively. The results are discussed in relation to flooding tolerance of these species.     

Expression of the T antigen on a T-lymphoid cell line, SupT1

We have measured glycosyltransferase activities of SupT1 cells, a T-lymphoid cell line shown to react with autoantibodies in the sera of many HIV patients. Since considerable -N-acetylgalactosaminyl-transferase and 1, 3 galactosyl-transferase activities were found in SupT1 cells, at least the O-glycan core 1 structure can probably be synthesized. FACS analysis using an anti-T monoclonal antibody showed expression of the T antigen (Gal 1-3 GalNAc). Glycoproteins with the T antigen were isolated by immunoprecipitation with the anti-T antibody from a SupT1 cell lysate labelled metabolically with³H-glucosamine and then analysed by SDS-PAGE. It was revealed that the precipitate contained a glycoprotein with a molecular weight corresponding to that of leukosialin. O-glycans were prepared from the immunoprecipitate by alkaline-borohydride treatment and then fractionated on Bio-Gel P-2, GalNAcOH and Gal-GalNAcOH being identifiedinter alia. These results suggest that an anti-T antibody may be included in the autoantibodies found in HIV-1 infected individuals.     

Covalent control of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase: insights into autoregulation of a bifunctional enzyme.

The hepatic bifunctional enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (6PF-2-K/Fru-2,6-P2ase), E.C. 2.7-1-105/E.C. 3-1-3-46, is one member of a family of unique bifunctional proteins that catalyze the synthesis and degradation of the regulatory metabolite fructose-2,6-bisphosphate (Fru-2,6-P2). Fru-2,6-P2 is a potent activator of the glycolytic enzyme 6-phosphofructo-1-kinase and an inhibitor of the gluconeogenic enzyme fructose-1,6-bisphosphatase, and provides a switching mechanism between these two opposing pathways of hepatic carbohydrate metabolism. The activities of the hepatic 6PF-2-K/Fru-2,6-P2ase isoform are reciprocally regulated by a cyclic AMP-dependent protein kinase (cAPK)-catalyzed phosphorylation at a single NH2-terminal residue, Ser-32. Phosphorylation at Ser-32 inhibits the kinase and activates the bisphosphatase, in part through an electrostatic mechanism. Substitution of Asp for Ser-32 mimics the effects of cAPK-catalyzed phosphorylation. In the dephosphorylated homodimer, the NH2- and COOH-terminal tail regions also have an interaction with their respective active sites on the same subunit to produce an autoregulatory inhibition of the bisphosphatase and activation of the kinase. In support of this hypothesis, deletion of either the NH2- or COOH-terminal tail region, or both regions, leads to a disruption of these interactions with a maximal activation of the bisphosphatase. Inhibition of the kinase is observed with the NH2-truncated forms, in which there is also a diminution of cAPK phosphorylation to decrease the Km for Fru-6-P. Phosphorylation of the bifunctional enzyme by cAPK disrupts these autoregulatory interactions, resulting in inhibition of the kinase and activation of the bisphosphatase. Therefore, effects of cyclic AMP-dependent phosphorylation are mediated by a combination of electrostatic and autoregulatory control mechanisms.     

The extracellular space and blood-eye barrier in an insect retina: An ultrastructural study

Summary (1) The distribution of the extracellular space (ECS) in the outer part of the locust compound eye has been mapped with lanthanum and ruthenium red, applied to the retina. (2) In the photoreceptor zone, about 2.4% of the volume is ECS, in agreement with radiotracer and electrical estimates. Of this ECS, about 70% lies in lacunae between ommatidia, but only 1–2% adjacent to the photosensitive rhabdom. The lacunae are filled with material which binds applied tracers, and are thought to be structural spaces. (3) It has been suggested several times that such a small cation pool is insufficient to sustain more than a few large photoresponses, but this is shown to be incorrect. Enough Na⁺ lies within the rhabdomal ECS and within rapid diffusional access to it, to impose no immediate limitation. (4) The palisade vacuoles surrounding the rhabdom are intracellular, and are typical of light as well as dark-adapted eyes. (5) Tracers fail to penetrate more than about 30 m into the axon zone, in agreement with electrical, dye and radiotracer indications of a blood-eye barrier near this point. Septate and gap junctions between glial membranes proliferate at this level, the lacunae disappear, and the axonal clefts narrow, but no tight junctions were seen. Comparison is made with the barrier around the nerve cord. (6) The secondary pigment cells in the retina may function as osmotic/ionic buffers, in conjunction with the blood-eye barrier.     

Rapid separation of the plastid,mitochondrial, and cytoplasmic fractions from intact leaf protoplasts of Avena

Purified intact protoplasts were isolated from etiolated and greening leaves of Avena sativa. They were ruptured by forcing them through a 20-m aperture nylon net and immediately thereafter fractionated into a pure pellet of plastids (well above 70% of total plastids), a layer of mitochondria only slightly contaminated by other cellular constituents (about 50% of total mitochondria), and a cytoplasmic supernatant. This was achieved within 60 s by an integrated method of homogenation of protoplasts and centrifugal filtration of the homogenate on a gradient of silicone oils, contained together with the nylon net in 450 l microtubes, and verified by comparing the levels of activity of specific markers within the three fractions obtained. With appropriate modifications to immediately quench metabolic reactions within the fractions, this method allows the determination of metabolite levels within plastids, mitochondria, and the cytoplasmic compartment of intact protoplasts. The applicability of this technique is demonstrated by the determination of ATP in the plastids, mitochondria, and the cytoplasm of protoplasts obtained from etiolated and greening primary leaves of Avena. The levels of ATP, corrected for contamination of the fractions by each other, exhibit a pronounced transient increase during greening, especially within the cytoplasm.Abbreviations BSA bovine serum albumin - Cyt c cytochrome c - EDTA ethylenediamine tetraacetic acid - HEPES N-2-hydroxyethyl-piperazine-N-2-ethane sulphonic acid - MES 2(N-morpholino) ethane sulphonic acid - PGA 3-phosphoglyceric acid - PEP phosphoenol pyruvic acid - RuBP ribulose-1.5-bis-phosphate