Striking Effects of Storage Buffers on Apparent Half-Lives of the Activity of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> Arylsulfatase

To obtain the label enzyme for enzyme-linked-immunoabsorbent-assay of two components each time in one well with conventional microplate readers, molecular engineering of Pseudomonas aeruginosa arylsulfatase (PAAS) is needed. To compare thermostability of PAAS/mutants of limited purity, effects of buffers on the half-activity time (t _0.5) at 37 °C were tested. At pH 7.4, PAAS showed non-exponential decreases of activity, with the apparent t _0.5 of ~6.0 days in 50 mM HEPES, but ~42 days in 10 mM sodium borate with >85 % activity after 15 days; protein concentrations in both buffers decreased at slower rates after there were significant decreases of activities. Additionally, the apparent t _0.5 of PAAS was ~14 days in 50 mM Tris–HCl, and ~21 days in 10 mM sodium phosphate. By sodium dodecyl-polyacrylamide gel electrophoresis, the purified PAAS gave single polypeptide; after storage for 14 days at 37 °C, there were many soluble and insoluble fragmented polypeptides in the HEPES buffer, but just one principal insoluble while negligible soluble fragmented polypeptides in the borate buffer. Of tested mutants in the neutral borate buffer, rates for activity decreases and polypeptide degradation were slower than in the HEPES buffer. Hence, dilute neutral borate buffers were favorable for examining thermostability of PAAS/mutants.     

A phloem-specific,lectin-like protein is located in pine sieve-element plastids by immunocytochemistry

Two co-purifying phloem polypeptides of 24 and 25 kilodaltons (kDa) were isolated from homogenates of Pinus sabiniana Dougl. phloem by differential centrifugation, selective solubilization and electrophoresis, and rabbit antibodies raised against them. The antisera were found to be specific for doublet bands between 23 and 25 kDa in Western blots of whole phloem extracts of Pinus species; no xylem polypeptides were labelled, nor did labelling occur in blots of phloem extracts from other genera in the Pinaceae. Solubilized phloem polypeptides bind strongly to chitin (oligomeric N-acetylglucosamine) columns and are sensitive to thiol reagents, both characteristics which relate them to phloemspecific lectins isolated from angiosperm species (C. Allen, 1979, Biochem. J. 183, 133–137; A.K. Gietl et al., 1979, Planta 144, 367–371). Fluorescence microscopy and immuno-gold electron microscopic cytochemistry demonstrated antigenic sites specifically associated with protein crystals peculiar to the sieve-element plastids of the Pinaceae.Abbreviations DAB diamino benzidine tetrachloride - FITC fluorescein isothiocyanate - kDa kilodalton - PBS phosphate-buffered saline - PP phloem polypeptide(s) - SDS-PAGE Sodium dodecyl sulphate-polyacrylamide gel electrophoresis - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

Electrophoretic comparison of polypeptides from enriched plasma membrane fractions from developing soybean roots

The polypeptide complement of enriched soybean (Glycine max [L.] Merr. cult. wells) root plasma membrane fractions was studied by two-dimensional gel electrophoresis. Good resolution was obtained when polypeptides were solubilized in sodium dodecyl sulfate and when butylated hydroxytoluene was included in the vesicle isolation and solubilization media. The pattern obtained on the two-dimensional slab gel for root plasma membrane was characteristic for that membrane. The polypeptide complements from mitochondrial membranes and from enriched fractions of three other endomembrane components were solubilized and electrophoresed for comparison. Each membrane preparation was identifiable on the basis of its characteristic electrophoretogram. Electrophoresis of protein solubilized from plasma membrane fractions isolated from meristematic and mature root tissue revealed both qualitative and quantitative differences in the respective protein complements.     

Boophilus microplus: characterization of larval phosphomonoesterases and isolation of subcellular fractions with high phosphatase activity.

Phosphomonoesterase activity was determined for a 115,000g pellet and soluble fractions resulting from a subcellular fractioning of a homogenate of larval Boophilus microplus. Both fractions showed maximum phosphatase activity at pH 5.5 and 10. Acid phosphatase (EC 3.1.3.2) activity was found to be greatest in the soluble fraction. When the reaction rate was plotted against homogenate concentration, the soluble acid phosphatase deviated from the linear relationship. For both fractions different thermostability patterns were obtained, inactlvation beginning for the alkaline phosphatase (EC 3.1.3.1) at 45–55 C. When the effect of substrate concentration on activity was studied, deviations from the typical hyperbolic behavior were observed. Homogenization of larvae with 5 mm EDTA buffer failed to yield a low-speed pellet with high alkaline phosphatase activity, as it is expected if absorptive structures sediment. Moreover, total alkaline phosphatase activity recovered by this method is significantly lower than activity recovered when homogenization is carried out without EDTA. Alternately, homogenization with 10 mM Tris buffer and 0.25 M sucrose gave 27,000g and 115,000g fractions with high phosphatase activity when fractioned by centrifugation. Alkaline treatment of the 115,000g fraction with 10 mM Tris buffer, pH 7.8, failed to separate endoplasmic reticulum contaminants without loss of phosphatase activity. When the 115,000g fraction was centrifuged in a sucrose density gradient, two activity peaks, coincident for both acid and alkaline phosphatases, were obtained. Antigenic analysis showed the existence of similar antigenic determinants in both peaks “immunologically” presented in different ways.     

Comparison of soluble and membrane polypeptides ofSynechocystis 6308 by two-dimensional electrophoresis

Two-dimensional electrophoresis was carried out on fractions of the cyanobacteriumSynechocystis 6308 (ATCC 27150). Phycobilisomes isolated fromSynechocystis in 0.75M potassium phosphate buffer, pH 6.8, (KPi) plus Triton X-100 showed 5 prominent polypeptides when examined by two-dimensional electrophoresis. Ultracentrifugation of cells broken in KPi lacking Triton yielded three fractions, a membrane-containing pellet, a green supernatant, and a less dense yellow supernatant. The three fractions yielded a total of 272 polypeptides visualized by silver staining of two-dimensional gels. Fourteen polypeptides were found only in the yellow fraction, 14 polypeptides were found only in the green fraction, and 16 polypeptides were found only in the membrane fraction; 23 polypeptides were found in all three fractions. The crude Triton-containing KPi extract contained 62, and a 50 mM HEPES extract contained 55, of the 272 polypeptides visualized in these fractions. Two-dimensional electrophoresis combined with subcellular fractionation may be useful tools for examining changes in polypeptide composition caused by nitrogen starvation.     

Seasonal dynamics of phloem and xylem formation in silver fir and Norway spruce as affected by drought

The dynamics of phloem growth ring formation in silver fir (Abies alba Mill.) and Norway spruce (Picea abies Karst.) at different sites in Slovenia during the droughty growing season of 2003 was studied. We also determined the timing of cambial activity, xylem and phloem formation, and counted the number of cells in the completed phloem and xylem growth rings. Light microscopy of cross-sections revealed that cambial activity started on the phloem and xylem side simultaneously at all three plots. However, prior to this, 1–2 layers of phloem derivatives near the cambium were differentiated without previous divisions. The structure of the early phloem was similar in silver fir and Norway spruce. Differences in the number of late phloem cells were found among sites. Phloem growth rings were the widest in Norway spruce growing at the lowland site. In all investigated trees, the cambium produced 5–12 times more xylem cells than phloem ones. In addition, the variability in the number of cells in the 2003 growth ring around the stem circumference of the same tree and among different trees was higher on the xylem side than on the phloem side. Phloem formation is presumably less dependent on environmental factors but is more internally driven than xylem formation.     

Cellulase and cell differentiation in Acer pseudoplatanus

Summary Homogenates of differentiating xylem and phloem tissue have higher cellulase activities than cambial samples; the highest activity is always found in phloem. Callus tissue, in which no vascular differentiation occurs, contains only low cellulase activity. The results suggest that cellulase is involved in vascular differentiation. Different pH optima of cellulase activity were found: in cambium, xylem and phloem tissue, cellulase activity with an optimum at about pH 5.9 is predominantly membrane-bound; it is sedimentable at 100,000 g and releasable by Triton X-100. The same may be true of activity with an optimum at pH 5.3. Phloem tissue also contains a soluble, cytoplasmic cellulase of high activity at pH 7.1, and xylem tissue contains cytoplasmic cellulase with an optimum at pH 6.5. Low cellulase activity with a pH optimum similar to that of xylem homogenates was found in xylem sap. Cellulase activity in abscission zones increases greatly just before leaf abscission. Abscission zone cellulase has two pH optima, et 5.3 and 5.9; both activities are increased by Triton treatment of homogenates. The possible existence of several different cellulases forming part of a cellulase complex, and the rôle of the enzymes in hydrolysing wall material during cell differentiation are discussed.     

Recycling efficiency in hydrogenase uptake positive strains of Rhizobium leguminosarum

Essentially chlorophyll-free preparations of mitochondria from different tissues of the same plant can be obtained by a combined three step preparation procedure involving differential centrifugation, partition in aqueous polymeric two-phase system and centrifugation in a Percoll gradient. The polypeptide patterns of mitochondria from photosynthetic (leaves) and non-photosynthetic (petioles and roots) tissue from spinach were compared by use of SDS-electrophoresis.
About 35 polypeptides were found in leaf mitochondria with molecular weights from 14 to 103 kdalton. The polypeptide patterns of the membrane fractions and matrix fractions showed great differences. The membrane fractions contained significantly more polypeptide bands than the matrix fractions. The polypeptide patterns of mitochondria from photosynthetic and non-photosynthetic tissues showed some striking differences. The 15.9, 41.7, 50.7 and 101 kdalton polypeptides were clearly detected in leaf mitochondria but these polypeptides were not found or found in only small amounts in petiole and root mitochondria. The differences were mainly associated with the matrix fractions. Staining with 3,3',5,5'-tetramethylbenzidine and hydrogen peroxide for heme containing polypeptides showed that the polypeptides which differ do not contain heme.     

Direct and indirect measurements of Phloem turgor pressure in white ash

Direct determinations and indirect calculations of phloem turgor pressure were compared in white ash (Fraxinus americana L.). Direct measurements of trunk phloem turgor were made using a modified Hammel-type phloem needle connected to a pressure transducer. Turgor at the site of the direct measurements was calculated from the osmotic potential of the phloem sap and from the water potential of the xylem. It was assumed that the water potentials of the phloem and xylem were close to equilibrium at any one trunk location, at least under certain conditions. The water potential of the xylem was determined from the osmotic potential of xylem sap and from the xylem tension of previously bagged leaves, measured with a pressure chamber. The xylem tension of bagged leaves on a branch adjacent to the site of the direct measurements was considered equivalent to the xylem tension of the trunk at that point. While both the direct and indirect measurements of phloem turgor showed clear diurnal changes, the directly measured pressures were consistently lower than the calculated values. It is not clear at present whether the discrepancy between the two values lies primarily in the calculated or in the measured pressures, and thus, the results from both methods as described here must be regarded as estimates of true phloem turgor.     

白鲜营养器官解剖结构及其与白鲜碱的积累关系

应用植物解剖学、组织化学及植物化学方法对白鲜营养器官根、茎、叶的结构及其生物碱的积累进行了研究。结果显示:(1)白鲜根的次生结构以及茎和叶的结构类似一般双子叶植物;白鲜多年生根主要由周皮、次生韧皮部、维管形成层以及次生木质部组成,根次生韧皮部中可见大量的淀粉、草酸钙簇晶、韧皮纤维以及油细胞;茎由表皮、皮层、维管组织和髓组成;叶由表皮、栅栏组织、海绵组织和叶脉组成;在茎和叶初生韧皮部的位置均分布有韧皮纤维,在叶表皮上分布有头状腺毛和非腺毛;在茎和叶紧贴表皮处分布有分泌囊。(2)组织化学分析结果显示:在白鲜多年生根中,生物碱类物质主要分布在周皮、次生韧皮部、维管形成层和木薄壁细胞中;在茎中,生物碱主要分布在表皮、皮层、韧皮部、木薄壁细胞及髓周围薄壁细胞中;在叶中,生物碱主要分布在表皮细胞、叶肉组织和维管组织的薄壁细胞;此外在分泌囊和头状腺毛中亦含有生物碱类物质。(3)植物化学结果显示,秦岭产白鲜根皮/白鲜皮、根木质部、茎和叶中白鲜碱含量分别为0.041%、0.012%、0.004%和0.002%,其中木质部中白鲜碱含量和其他部分地区白鲜皮中白鲜碱含量类似。研究表明,在秦岭产白鲜营养器官中,除根皮/白鲜皮外,在根木质部亦含有大量的白鲜碱,且在茎和叶中亦含有一定的白鲜碱,具有潜在的开发利用价值。     

Variation in wood density and anatomy in a widespread mangrove species

Wood density is an important plant trait that influences a range of ecological processes, including resistance to damage and growth rates. Wood density is highly dependent on anatomical characteristics associated with the conductive tissue of trees (xylem and phloem) and the fibre matrix in which they occur. Here, we investigated variation in the wood density of the widespread mangrove species Avicennia marina in the Exmouth Gulf in Western Australia and in the Firth of Thames in New Zealand. We assessed how variation in xylem vessel size, fibre wall thickness and proportion of phloem within the wood contributed to variation in wood density and how these characteristics were linked to growth rates. We found the wood density of A. marina to be higher in Western Australia than in New Zealand and to be higher in taller seaward fringing trees than in scrub trees growing high in the intertidal. At the cellular level, high wood density was associated with large xylem vessels and thick fibre walls. Additionally, wood density increased with decreasing proportions of phloem per growth layer of wood. Tree growth rates were positively correlated with xylem vessel size and wood density. We conclude that A. marina can have large xylem vessel sizes and high growth rates while still maintaining high wood density because of the abundance and thickness of fibres in which vessels are found.     

Studies on the polypeptide composition of the cyanobacterial oxygen-evolving complex

Various approaches have been used to investigate the polypeptides required for oxygen evolution in cyanobacteria, in particular the thermophile Phormidium laminosum. Antibodies against the extrinsic 33 kDa protein from spinach Photosystem II cross-reacted clearly in immunoblotting experiments with a corresponding polypeptide in isolated thylakoids and Photosystem II particles from P. laminosum and with whole-cell homogenates of three species of cyanobacteria (Phormidium laminosum, Synechococcus leopoliensis and Anabaena variabilis). In contrast, no cyanobacterial proteins reacted with antibodies against the 23 and 16 kDa proteins of spinach Photosystem II. The lack of cross-reactivity and the absence of these polypeptides from highly active Photosystem II particles of Phormidium laminosum strongly suggest that cyanobacteria do not contain polypeptides corresponding to these two chloroplast proteins. Treatment of P. laminosum Photosystem II particles with 0.8 M alkaline Tris, 1 M NaCl, CaCl₂ or MgCl₂ inhibited O₂ evolution, and quantitatively removed a 9 kDa polypeptide from the particles. None of these treatments removed comparable amounts of the 33 kDa polypeptide, and only Tris treatment removed manganese. The release of the 9 kDa polypeptide upon NaCl treatment correlated well with the deactivation at the donor side of Photosystem II. A direct connection between the 33 kDa polypeptide and O₂ evolution was established by the finding that trypsin treatment digested this polypeptide and inhibited O₂ evolution in parallel.     

Studies on the preparation of rat liver plasma membrane fractions and on their polypeptide patterns

Plasma membrane and bile canalicular membrane fractions were prepared from rat liver using NaHCO3, NaHCO3--CaCl2, and K2HPO4-KH2PO4 buffers (all at pH 7.4). The amount (expressed as milligrams protein per gram liver) of plasma membrane fraction exceeded the amount of bile canalicular membrane fraction using each of these three media; the use of NaHCO3-CaCl2 afforded a substantially higher yield of both types of membranes. The two membrane fractions exhibited complex patterns of polypeptides (greater than 30) on sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis. Several reproducible differences in polypeptide patterns were observable between the two membrane fractions; in particular, components possibly corresponding to the heavy chain of myosin and to action were prominent in the bile canalicular membrane fraction. The effects of incubation in the above three buffers and in Tris--HCl (pH 7.4) on the polypeptide patterns of both types of membrane were studied. Many polypeptides were released from each type of membrane in all of these media. Differential effects on the polypeptide patterns of either type of membrane fraction were observed among the various buffers. In terms of minimizing loss of polypeptides, in general, NaHCO3--CacCl2 appeared to be the best buffer and Tris--HCl the worst buffer. The significance of these results for the preparation and storage of liver cell plasma membrane fractions is briefly discussed.     

EFFECTS OF TREE AGE ON SECONDARY XYLEM AND PHLOEM ANATOMY IN STEMS OF GREAT BASIN BRISTLECONE PINE (PINUS LONGAEVA)

Xylem and phloem tissue samples were collected from various-aged Great Basin bristlecone pine (Pinus longaeva D. K. Bailey) stems in southern Utah and southeastern California to determine whether the vascular cambia of older trees produce fewer xylem rays, shorter-lived xylem and phloem ray cells, fewer phloem sieve cells, and a thinner phloem. Increment cores were examined to determine whether ‘aged’ cambia produced narrower tracheids that might reduce water translocation. Sapwood thickness was measured and sapwood growth layers were counted on these cores. Regression and Classification and Regression Tree (CART) analyses of sample data found no age-related changes in cambial products. Phloem and xylem production appeared normal at all ages, with no evidence of cambial malfunction.     

Xylem and Phloem Transport of Mineral Nutrients from Solanum tuberosum Roots

The xylem and phloem transport of mineral elements from stemnodal roots to the stem and stolon of growing potato (Solanumtuberosum L. cv. ‘Russet Burbank’) plants was investigated.Adventitious roots, originating from below-ground nodes of thestem of potato seedlings, were exposed to solutions of SrCI₂or MnSO₄. Relative elemental concentrations were measured inthe conductive tissues using energy dispersive X-ray analysis.After a 5 h daylight uptake period, Sr (a Ca-transport analogue)levels were elevated in the stem xylem tissue, but Sr did notincrease in the stem phloem, nor was it present in either ofthe conductive tissues of stolons located 1–2 nodes abovethe treated roots. In contrast, elevated levels of Cl, S, andMn were found in stolon xylem and phloem tissue during the sameperiod. The absence of Sr in the stolon after 5 h suggests thatno xylem flow into the stolon occurred during the uptake periodand, furthermore, phloem flow is responsible for the transportof the Cl, S, and Mn into the stolon. Elevated levels of thesemobile nutrients in the xylem of the stolon were attributedto xylem-to-phloem transfer in the stem or leaves, transportto the stolon in the phloem, and phloem-to-xylem transfer inthe stolon. During a 19 h uptake period, some Sr was observedin the phloem tissue of the stem, demonstrating slow exchangeof Sr with sieve elements or proximal phloem parenchyma andcompanion cells. Key words: Calcium, manganese, X-ray analysis     

ISOLATION OF NERVE ENDINGS FROM THE POSTERIOR PITUITARY GLAND : Electron Microscopy of Fractions Obtained by Centrifugation

Bovine posterior pituitary glands were homogenized in 10 per cent sucrose and fractionated by differential centrifugation. The following centrifugation procedure resulted in the most satisfactory separation: 1000 g for 15 minutes—nuclei, connective tissue, basement membranes with associated endothelium, giant nerve endings, and whole pituicytes; 4200 g for 15 minutes—free nerve endings, including Herring bodies; 17,000 g for 15 minutes—mitochondria; 68,000 g for 15 minutes—neurosecretory granules. Electron microscopic examination was carried out on whole tissue and on the isolated fractions. Isolated nerve endings were examined also by negative staining techniques. Isolated nerve endings retain an apparently normal complement of mitochondria, neurosecretory granules, and microvesicles ("synaptic" vesicles). The free nerve endings closely resemble those observed in sections of intact posterior pituitary tissue. Free microvesicles were not observed in any of the fractions isolated and apparently sediment at centrifugal forces higher than those employed in this study.     

Wood Anatomy and the Development of Interxylary Phloem of Ipomoea hederifolia Linn. (Convolvulaceae)

In Ipomoea hederifolia Linn., stems increase in thickness by forming successive rings of cambia. With the increase in stem diameter, the first ring of cambium also gives rise to thin-walled parenchymatous islands along with thick-walled xylem derivatives to its inner side. The size of these islands increases (both radially and tangentially) gradually with the increase in stem diameter. In pencil-thick stems, that is, before the differentiation of a second ring of cambium, some of the parenchyma cells within these islands differentiate into interxylary phloem. Although all successive cambia forms secondary phloem continuously, simultaneous development of interxylary phloem was observed in the innermost successive ring of xylem. In the mature stems, thick-walled parenchyma cells formed at the beginning of secondary growth underwent dedifferentiation and led to the formation of phloem derivatives. Structurally, sieve tube elements showed both simple sieve plates on transverse to slightly oblique end walls and compound sieve plates on the oblique end walls with poorly developed lateral sieve areas. Isolated or groups of two to three sieve elements were noticed in the rays of secondary phloem. They possessed simple sieve plates with distinct companion cells at their corners. The length of these elements was more or less similar to that of ray parenchyma cells but their diameter was slightly less. Similarly, in the secondary xylem, perforated ray cells were noticed in the innermost xylem ring. They were larger than the adjacent ray cells and possessed oval to circular simple perforation plates. The structures of interxylary phloem, perforated ray cells, and ray sieve elements are described in detail.     

Ascorbic acid and development of xylem and phloem cells in the pine trunk

Changes in the levels of ascorbic acid (AA), its oxidized form, dehydroascorbic acid (DHA), and uronic acids as initial precursors for the AA synthesis were studied as related to the degree of xylem and phloem cell development in the course of early and late wood formation in the trunks of Scots pine (Pinus sylvestris L.). The cells of mature and conducting phloem, cambial zone, differently developed cells in the zones of cell enlargement and maturation were obtained by successive scraping tissue layers from trunk segments of 20–25-year-old trees; tissue identification was checked anatomically and histochemically. The contents of compounds tested were calculated per dry weight and per cell basis. We found great differences in the contents of AA and DHA and also in their ratio in dependence of the wood type developing in the pine trunks during growth period and on the stage of differentiation of xylem and phloem cells. Changes in the AA content during xylem cell differentiation were accompanied by changes in the content of uronic acids. The amounts of AA, DHA, and uronic acids were the highest at the stage of early lignification and reduced with tracheid maturation. The AA to DHA ratio changed differently in the course of early and late xylem lignification. It reduced from the start of lignification to the formation of early mature xylem and, in contrast, increased in mature late wood; this indicates a difference in the level of redox processes in these tissues.     

Isolation and in vitro assembly of the components of the outer S layer of Lampropedia hyalina.

The outermost component of the S layer of Lampropedia hyalina, the punctate layer, is assembled onto an inner perforate layer. The punctate layer is composed of long, tapered cylindrical units centered on p6 symmetry axes and connected by six fine linking arms, joining at the axis of threefold symmetry to create a hexagonal layer with a lattice constant of 25.6 +/- 0.5 nm (J. A. Chapman, R. G. E. Murray, and M. R. J. Salton, Proc. R. Soc. London Ser. B 158:498-513, 1963; R. G. E. Murray, Can. J. Microbiol. 9:593-600, 1963). Extraction of cell envelopes with 100 mM Tris buffer (pH 8) containing 2% deoxycholate resulted in the release of several proteins, but left the S layers intact. The punctate layer was then extracted with 3 M guanidine hydrochloride or 6 M urea, leaving the perforate layer intact. This treatment led to the release of three polypeptides with molecular weights of 60,000, 66,000, and 240,000 (60K, 66K, and 240K polypeptides). These three polypeptides reassembled on the perforate layer as a template to form the S-layer complex or self-assembled to form the punctate layer alone after dialysis of the extract against 50 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer (pH 7.5) containing 10 mM CaCl2. The self-assemblies were composed of a 240K polypeptide and a 60K polypeptide. The 240K and 60K polypeptides were separated by column chromatography and examined by electron microscopy. The 240K polypeptide appeared in negative stain as a long, flexible structure and assembled into loose arrays with sixfold symmetry with obvious Y-shaped linking elements, while fractions containing both the 60K and 240K polypeptides showed assemblies closely resembling the punctuate layer. Immunoelectron microscopy was used to confirm the presence of both the 60K and 240K polypeptides as components of the punctuate layer.