Cholesteric-like crystal analogs in glucuronoxylan-rich cell wall composites: Experimental approach of acellular re-assembly from native cellulosic suspension

Summary Many plant cell walls are constructed according to a helicoidal pattern that is analog to a cholesteric liquid crystal order. This raises the question whether the wall assembly passes through a true but temporary liquid crystal state. The paper focuses on experiments performed from aqueous suspensions of extracted quince slime, i.e., a cellulose/glucuronoxylan wall composite that presents a helicoidal order when observed in situ, within the enlarged periplasm of the seed epidermal cells. Experiments carried out in acellular conditions showed that a spontaneous reassociation into a helicoidal order can be obtained from totally dispersed suspensions. The ultrastructural aspect of the reassembled mucilage suspension was different according to the resin used (LR White or nanoplast, a water-soluble melamin resin). It was always typically polydomain, and when an order was visible it was cholesteric-like and similar to the in situ native organization. Transition states with many imperfections expressed the difficulty of the system to reassemble in the absence of constraining surfaces. The possible intervention of glucuronoxylan (GX) in the ordered assembly of the microfibrils was checked by: (1) progressive extraction of GX by trifluoroacetic acid (TFA). The extraction was associated to a control of the fraction by analysis of uronic acid contents and observation at the electron microscope level. Extraction of GX provoked the formation of a flocculent mass, the flocculation being more intense when the TFA was more concentrated; (2) progressive change of pH in order to analyze the influence of pH on flocculation. Low pH (ca. pH 3) led also to a flocculation of the suspension, but the floc was reversibly lost after dialysis against distilled water. The results indicate the antifloc role of the GX due to the anionic charges carried by the side-chains. However, the function of GX as helper twisting agent in the cholesteric-like reassembly must not be ruled out.     

Purification and Kinetic Properties of Serine Acetyltransferase Free of O-Acetylserine(thiol)lyase from Spinach Chloroplasts

Serine acetyltransferase, a key enzyme in the L-cysteine biosynthetic pathway, was purified over 300,000-fold from the stroma of spinach (Spinacia oleracea) leaf chloroplasts. The purification procedure consisted of ammonium sulfate precipitation, anion-exchange chromatography (Trisacryl M DEAE and Mono Q HR10/10), hydroxylapatite chromatography, and gel filtration (Superdex 200). The purified enzyme exhibited a specific activity higher than 200 units mg-1 and a subunit molecular mass of about 33 kD upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Moreover, the purified serine acetyltransferase appeared to be essentially free of O-acetyleserine(thiol)lyase, another enzyme component in the L-cysteine biosynthetic pathway. A steady-state kinetic analysis indicated that the mechanism of the enzyme-catalyzed reaction involves a double displacement. The apparent Km for the two substrates, L-serine and acetyl-coenzyme A, were 2.29 [plus or minus] 0.43 and 0.35 [plus or minus] 0.02 mM, respectively. The rate of L-cysteine synthesis in vitro was measured in a coupled enzyme assay using extensively purified O-acetylserine(thiol)lyase and serine acetyltransferase. This rate was maximum when the assay contained approximately a 400-fold excess of O-acetylserine(thiol)lyase over serine acetyltransferase. Measurements of the relative level of O-acetylserine(thiol)lyase and serine acetyltransferase activities in the stroma indicated that the former enzyme was present in much larger quantities than the latter. Thus, the activity ratio for these two enzymes [O-acetylserine(thiol)lyase activity/serine acetyltransferase activity] measured in the stromal protein extract was 345. This strongly suggested that all the O-acetylserine(thiol)lyase and serine acetyltransferase activities in the stroma are involved in bringing a full synthesis of L-cysteine in the chloroplast.     

Gene transfer to inflorescence and flower meristems using ballistic micro-targeting

Direct gene transfer to floral meristems could contribute to cell-fate mapping, to the study of flower-specific genes and promoters, and to the production of transgenic gametes via the transformation of sporogenic tissues. Despite the wide potential of its applications, direct gene transfer to floral meristems has not been achieved so far because of the lack of suitable technology. We show in this paper that ballistic micro-targeting is the technique of choice for this purpose, and in this way, we were able to transfer genes efficiently into excised wheat immature spikes. Particle size was adjusted for optimal penetration into the L1 and L2 cell layers of the spikes with limited cell damage. Spikes at different developmental stages were shot either with a plasmid containing two genes involved in anthocyanin biosynthesis or with a plasmid bearing the uidA (-glucuronidase) gene. The transient expression of these marker genes was observed in the different developmental stages tested and in cells of both the L1 and the L2 layers. The transient expression of the uidA gene was significantly increased when the sucrose concentration in the culture medium was increased from 0.06 to 0.52 M. At the highest concentration, 100% of the targeted spikes expressed the uidA gene, with an average of 69 blue cells per spike. Twelve days after microtargeting, multicellular sectors showing transgene expression and containing up to 17 cells were found in 85% of the shot immature inflorescences. This indicated that targeted cells survived particle bombardment. Sectors were found in primordia of both vegetative and reproductive organs.     

Purification and characterization of CMP-N-acetylneuraminic acid hydroxylase from pig submandibular glands

N-Glycoloylneuraminic acid (Neu5Gc) is synthesized as its CMP-giycosideby the action of CMPN-acetylneuramlnic acid (CMP-Neu5Ac) hydroxylase.This enzyme is a soluble cytochrome bs-dependent monooxygenaseand has been purified to apparent homogeneity from pig submandibularglands by precipitation with N-cetyN,N,N-trimethylam-moniumbromide and fractionation on Q-Sepharose, Cibacron Blue 3GA-Agarose,Reactive Brown 10-Agarose, Hexyl-Agarose and Superose S.12.This procedure resulted in an 8960-fold purification of thehydroxylase with a recovery of 0.8%. The molecular mass of thisprotein was shown to be 65 kDa on SDS-PAGE and 60 kDa as determinedby gel filtration on Superose S.12, which suggests that theenzyme is a monomer. The purified CMP-Neu5Ac hydroxylase isactivated by FeSO₄ and inhibited by iron-binding reagents suchas o-phenanthroline, KCN, Tiron and ferro-zine. An apparentKm of 11 µM was determined for the substrate CMP-Neu5Acusing purified hydroxylase in the presence of Triton X-100-solubilizedmicrosomes. In a reconstituted system consisting of purifiedhydroxylase, cytochrome b₅, cytochrome b₅ reductase and catalase,an apparent K_m of 3 µM was measured. The apparent K_mforcytochrome b₅ in this system was 0.24 µM. Immunizationof a rabbit with enriched and purified hydroxylase led to anantiserum that inhibited CMP-Neu5Ac hydroxylase activity andreacted with the purified 65 kDa protein on a Western blot afterSDS-PAGE. Antibodies specific for this 65 kDa protein were isolatedand showed a strong reaction with the purified CMP-Neu5Ac hydroxylasefrom mouse liver after immunoblotting. Initial experiments withthis monospecific antibody suggest that the activity of thehydroxylase in a particular tissue correlates with the amountof immuno-reactive protein. cytochrome b₅ N-glcoloylneuraminic acid hydroxylase pig submandibular gland mucin sialic acid     

Modification of sialic acids by 9-O-acetylation is detected in human leucocytes using the lectin property of influenza C virus

Influenza C virus spike glycoprotein HEF specifically recognizesglycoconjugates containing 9-O-acetyl-N-acetylneuraminic acid.The same protein also contains an esterase activity. Takingadvantage of these two properties, influenza C virus was usedas a very sensitive probe for the detection of traces of 9-O-acetyl-N-acetylneuraminicacid in human leucocytes. The binding of influenza C virus toleucocyte glycoproteins and gangliosides separated by sodiumdodecyl sulphate–polyacrylamide gel electrophoresis andthin-layer chromatography, respectively, was assayed using achromogenic esterase substrate. In this way, glycoproteins ofB-lymphocytes and T-lymphocytes were found to contain 9-O-acetylatedsialic acids. Of the various 9-O-acetylated gangliosides detected,one had the characteristics of 9-O-acetylated G^D3. The identificationof 9-O-acetylated sialic acids on distinct glycoproteins andglycolipids should be helpful in assigning a physiological roleto this sugar. O-acetylation gangliosides influenza C virus lymphocytes sialic acids     

Chromosomal localization of the human histamine H1-receptor gene

We have assigned the human histamine H₁-receptor gene to chromosome 3 by Southern blot analysis of a chromosome mapping panel constructed from humanhamster somatic cell hybrids. This assignment was confirmed by in situ hybridization on metaphase chromosomes and involved bands 3p14–p21.     

Cloning and nucleotide sequence analysis of the Lactobacillus delbrueckii ssp. lactis DSM7290 cysteine aminopeptidase gene pepC

Abstract A genomic library of Lactobacillus delbrueckii ssp. lactis DSM7290 in the low copy number vector pLG339, was screened for the presence of peptidase genes. Using the chromogenic substrate gly-ala-β-naphthylamide, which is not a substrate for any of the recently cloned peptidases of DSM7290, and the multiple peptidase deficient Escherichia coli strain CM89, allowed the isolation of clones, which contained the equivalent hydrolytic activity. To identify genes encoding the conserved catalytic active site of cysteine proteases, partial nucleotide sequencing with a degenerate oligonucleotide was performed on recombinant plasmids isolated from such clones. This allowed to identify two out of nine clones to carry the Lactobacillus pepC gene. A total of 2026 nucleotides were determined, and sequence analysis revealed a gene with strong homology to the recently cloned Lb. helveticus (73.2%) and Lactococcus lactis (51.03%) pepC genes, and the derived protein showed homology with the active site of a large number of cysteine proteases. The predicted open reading frame consists of 449 codons, coding for a protein of 50 909 Da. The enzyme is functional and extremely overexpressed in E. coli .     

Variation and taxonomy in Hordeum depressum and in the H. brachyantherum complex (Poaceae)

The variation pattern in the perennial Hordeum brachyantherum complex and in the annual H. depressum are described. The diploid form of H. brachyantherum s. lat., endemic to California in USA, previously recognized as a separate species is here treated as a subspecies ( H. brachyantherum ssp. californicum ). Despite its restricted distribution it shows a considerable variation and overlap in morphology with the tetraploid ssp. brachyantherum , and no unambiguous determination based on morphology between the two tax a is possible. The tetraploid cytotype has a large distribution area in western North America and easternmost Asia and a very wide morphological variation. It has also a small disjunct distribution area in easternmost Canada. A single hexaploid population from California is referred to ssp. brachyantherum .     

Growth Hormone Secretion after Testosterone Administration to Men with Visceral Obesity

Visceral obese men were characterized by a decreased total GH secretion and diminished peak amplitude, size, and number. T-substitution was followed by elevation of IGF-I levels. The IGF-I increase correlated with the elevation of T-concentration, and was most pronounced in men with the lowest concentrations of free T from the outset. There were no detectable changes in total quantity, amplitude, size or number of peaks of GH secretion. Glucose, cholesterol and triglycerides as well as diastolic blood pressure decreased. There were no changes in thyroid or hematology variables. Visceral obesity in men has been reported to be characterized by low testosterone (T) and insulin-like growth factor I (IGF-I) concentrations, the latter suggesting a relative growth hormone (GH) deficiency. Since T and GH-secretions are interrelated, men with visceral obesity were substituted with T for 14 days, and diurnal secretion pattern of GH as well as IGF-I concentrations, and metabolic variables were followed. T-substitution of visceral obese men is followed by an elevation of IGF-I concentrations. It is suggested that this might be due either to minor, non-detectable increases in GH secretion, or to direct effects of T on IGF-I concentrations. The regulatory mechanisms by which T-administration are leading to metabolic and anthropometric improvements, might be direct effects of T, with or without mediation via GH secretion.     

The influence of catchment size on lake trophic status during the hemlock decline and recovery (4800 to 3500 BP) in southern Ontario lakes

We investigated the ecological effects of terrestrial ecosystem change during the hemlock decline and recovery (4,800–3,500 BP) on lake communities (diatoms and chrysophytes). This study specifically assessed the role of catchment area and slope in determining the magnitude of lake eutrophication during the hemlock decline by analyzing sediment cores from five alkaline, holomictic lakes in southeastern Ontario, Canada. The study lakes were similar in most limnological aspects, but differed widely in the relative sizes of their catchments. Diatoms were used to quantitatively infer past lake-water total phosphorus (TP) concentrations.All five lakes showed shifts in their algal communities during the hemlock decline, but most lakes exhibited only minor changes in trophic status. The magnitude of the limnological response appears to be related to catchment size and slope. Long Lake, Burridge Lake, and Gunter Lake possess the smallest catchments and exhibited the weakest responses to the hemlock decline. The catchment area of Flower Round Lake is considerably larger and steeper than these lakes, and was the only lake to show a marked eutrophication. Aulacoseira ambigua bloomed and diatom-inferred TP concentration increased by 14 µg 1^–1.Catchment slope appears to have influenced the type of material exported into the lakes. Lake basins draining catchments with gentle relief received proportionally greater amounts of organic matter, whereas steeper catchments supplied relatively greater proportions of mineral matter. Faster water flow associated with steeper catchment slope may have enhanced mineral erosionFollowing the hemlock decline, nutrient supplies to most of the study lakes were reduced. The period of forest recovery was associated with an 11 µg 1^–1 reduction in diatom-inferred lake-water TP concentration in Flower Round Lake, and algal populations decreased. Our results generally support the ecological theory of forest ecosystem development and secondary succession developed from long-term data collected at the Hubbard Brook Experimental Ecosystem.