Hydrolysis of steryl esters by a lipase (Lip 3) from Candida rugosa

A well-known lipase, Lip 3 of Candida rugosa, was purified to homogeneity from a commercial lipase preparation, using hydrophobic interaction and anion exchange chromatography. Lip 3, which has been reported to act on cholesteryl esters, was also found to be active on plant-derived steryl esters. Lip 3 had optimal activity at pH 5-7 and below 55 degrees C. It was able to hydrolyse steryl esters totally in a clear micellar aqueous solution. However, the action on a dispersed colloidal steryl ester solution was limited and only about half of the steryl esters were degraded. The degree of hydrolysis was not improved by addition of fresh enzyme. The composition of released fatty acids and sterols was, however, almost identical to that obtained by alkaline hydrolysis, showing that all the different steryl esters were hydrolysed equally and that none of the individual components were responsible for incomplete hydrolysis. Thus, it appeared that the physical state of the colloidal steryl ester dispersion limited the action of Lip 3. Wood resins contain both triglycerides and steryl esters among the hydrophobic components, which create problems in papermaking. The simultaneous enzymatic hydrolysis of triglycerides and steryl ester is therefore of considerable interest and Lip 3 is the first enzyme reported to act on both triglycerides and steryl esters.     

Variation of lignans in Norway spruce (Picea abies [L.] Karst.) knotwood: within-stem variation and the effect of fertilisation at two experimental sites in Finland

Lignan concentrations in Norway spruce (Picea abies [L.] Karst.) knotwood were studied in two long-term nitrogen fertilised experimental sites: a boreal site (66°5′N, 27°07′E) close to the Arctic tree line in northern Finland and a boreal site (61°10′N, 26°03′E) in southern Finland. Altogether 20 mature trees, representing five different size classes, were felled in the autumn 2002 and knots of the largest and smallest diameter in each whorl at the height of 4 m were analysed. Within-stem variation of knotwood lignans was studied at the 4 m intervals from stump height to the top from five control trees in the southern site. The following knotwood lignans were identified with GC-MS: hydroxymatairesinol (two isomers), secoisolariciresinol, α-conidendrin, α-conidendric acid, isohydroxymatairesinol, lariciresinol, lignan A, matairesinol, nortrachelogenin, todolactol A and isoliovil. The predominant lignan in Norway spruce knotwood was hydroxymatairesinol comprising approximately 77% of the total concentration of lignans. In mature trees in the southern site, the lignan concentrations were highest close to the starting point of the living branches. In the northern site, the total concentration of knotwood lignans was significantly higher (approximately 14% of the dry mass) than in the southern site (approximately 5.4% of the dry mass). In the northern site in control trees, a negative linear correlation was detected between branch diameter and hydroxymatairesinol concentration, but the similar correlation was not detected in fertilised trees or in the trees of the southern site. The possible reasons for higher hydroxymatairesinol concentrations in the northern knots were discussed.     

Selecting representative model micro-organisms

Background

The sexually transmitted disease, gonorrhea, is a serious health problem in developed as well as in developing countries, for which treatment continues to be a challenge. The recent completion of the genome sequence of the causative agent, Neisseria gonorrhoeae, opens up an entirely new set of approaches for studying this organism and the diseases it causes. Here, we describe the initial phases of the construction of an expression-capable clone set representing the protein-coding ORFs of the gonococcal genome using a recombination-based cloning system.

Results

The clone set thus far includes 1672 of the 2250 predicted ORFs of the N. gonorrhoeae genome, of which 1393 (83%) are sequence-validated. Included in this set are 48 of the 61 ORFs of the gonococcal genetic island of strain MS11, not present in the sequenced genome of strain FA1090. L-arabinose-inducible glutathione-S-transferase (GST)-fusions were constructed from random clones and each was shown to express a fusion protein of the predicted size following induction, demonstrating the use of the recombination cloning system. PCR amplicons of each ORF used in the cloning reactions were spotted onto glass slides to produce DNA microarrays representing 2035 genes of the gonococcal genome. Pilot experiments indicate that these arrays are suitable for the analysis of global gene expression in gonococci.

Conclusion

This archived set of Gateway^® entry clones will facilitate high-throughput genomic and proteomic studies of gonococcal genes using a variety of expression and analysis systems. In addition, the DNA arrays produced will allow us to generate gene expression profiles of gonococci grown in a wide variety of conditions. Together, the resources produced in this work will facilitate experiments to dissect the molecular mechanisms of gonococcal pathogenesis on a global scale, and ultimately lead to the determination of the functions of unknown genes in the genome.     

Knots in trees – A new rich source of lignans

Recent research in our group has revealed that knots, i.e. the branch bases inside tree stems, commonly contain 5–10% (w/w) of lignans. Norway spruce (Picea abies) knots contain as much as 6–24% of lignans, with 7-hydroxymatairesinol (HMR) as the predominant (70–85%) lignan. Some other spruce species also contain HMR as the main lignan, but some spruce species have also other dominating lignans. Most fir (Abies) species contain secoisolariciresinol and lariciresinol as the main lignans. Lignans occur also in knots of pines (Pinus spp.), although in lower amounts than in spruces and firs. Scots pine (Pinus silvestris) knots were found to contain 0.4–3% of lignans with nortrachelogenin as the main lignan. Lignans have been identified also in knots of some hardwoods, although flavonoids are more abundant in hardwoods. Knots are detrimental in the manufacture of pulp and paper and should preferably be removed before pulping. This is possible using a recently developed industrially applicable process called ChipSep. Recent research has also established novel synthetic routes to several lignans, such as matairesinol, secoisolariciresinol, lariciresinol and cyclolariciresinol, starting from hydroxymatairesinol by applying fairly straight-forward chemical transformations. We conclude that wood knots in certain spruce and fir species constitute the richest known source of lignans in nature. The lignans occur in knots in free form and are easily extracted by aqueous ethanol, or even by water. Not only HMR, but also other potentially valuable lignans, could be produced in a scale of hundreds of tons per year by extraction of knots separated from wood chips at pulp and paper mills.     

A mutation in aspergillus nidulans that blocks the transition from interphase to prophase

In order to develop a method for obtaining mitotic synchrony in aspergillus nidulans, we have characterized previously isolated heat-sensitive nim mutations that block the nuclear division cycle in interphase at restrictive temperature. After 3.5 h at restrictive temperature the mitotic index of a strain carrying one of these mutations, nimA5, was 0, but when this strain was subsequently shifted from restrictive to permissive temperature the mitotic index increased rapidly, reaching a maximum of 78 percent after 7.5 min. When this strain was examined electron-microscopically, mitotic spindles were absent at restrictive temperature. From these data we conclude that at restrictive temperature nimA5 blocks the nuclear division cycle at a point immediately preceding the initiation of chromosomal condensation and mitotic microtubule assembly, and upon shifting to permissive control over the initiation of microtubule assembly and chromosomal condensation in vivo through a simple temperature shift and, consequently, nimA5 should be a powerful tool for studying these processes. Electron-microscopic examination of spindles of material synchronized in this manner reveals that spindle formation, although very rapid, is gradual in the sense that spindle microtubule numbers increase as spindle formation proceeds.     

Comparison of triphenyltetrazolium chloride (TTC) staining versus detection of fibronectin in experimental myocardial infarction

Staining with triphenyltetrazolium chloride (TTC), although controversial, has frequently been used for the delineation of myocardial infarction. This study was performed further to explore the reliability of the TTC method. In 24-h experiments pigs were subjected to closed-chest occlusion of the left anterior descending coronary artery for 30, 60 or 90 min followed by reperfusion with or without superoxide dismutase (SOD) as an adjunct. One TTC-stained slice from each heart was stabilized by microwave irradiation, gelatin-embedded, frozen in hexane chilled with dry ice and cryosectioned. Serial sections were stained with antibodies against fibronectin in order to identify irreversibly injured myocytes and with van Gieson histologically to confirm the necrotic tissue. A close correspondence of the infarct size was found between TTC stained slices and anti-fibronectin stained sections. The infarct size in the van Gieson stained sections also showed good correspondence but the area of infarction tended to be larger. In the experimental group subjected to 30 min ischaemia and with SOD as an adjunct, the estimated infarcted area in the TTC stained slices was significantly smaller than the area estimated from the anti-fibronectin stained sections. In sections viewed in the light microscope an inverse pattern of TTC and anti-fibronectin staining was observed. It was confirmed at the light microscopic level that myocytes containing an abundance of TTC deposits lacked fibronectin whereas myocytes stained with antifibronectin in general lacked TTC staining except for a zone approximately 0.5 mm wide which was located at the intersection between damaged and surviving myocytes where small TTC deposits were present. The width of the stained zone did not differ among the experimental groups. Thus, differences in estimated infarct size by the three methods used reflect problems in correctly delineating the border between living and dead myocardium rather than an interference by SOD on TTC staining.     

A method for enzyme- and immunohistochemical staining of large frozen specimens

Summary The extracellular presence of adenosine polyphosphatase was investigated in a number of rat tissues. The enzyme was demonstrated in basement membranes of epithelial cells of duodenum, urinary bladder, tongue, choroid plexus, submandibular salivary gland, lung and kidney, as well as in basement membranes of capillaries in these tissues. Furthermore adenosine polyphosphatase was demonstrated on collagen fibrils and in the cytoplasm of fibroblasts of all investigated tissues. It appears that the presence of adenosine polyphosphatase in basement membranes is a widespread phenomenon. Since extracellular ADP and ATP are known to promote respectively platelet aggregation and inflammation, the presence of extracellular ADP and ATP-hydrolyzing activity might contribute to inhibit these processes.     

Some virus and virus-like diseases of tobacco,tomato, papaya,and rubber tree in vietnam and cambodia

In Vietnam a green strain of tobacco mosaic virus was isolated having TIP 89°C (10 min) and causing systemic necrosis in tobacoo ‘Xanthi-nc’ and sometimes also inDatura stramonium. In symptomless tomato plants an elongated virus belonging apparently to the Carlavirus group (NL 630 nm) was found. In papaya trees showing severe symptoms of mosaic and/or ringspot elongated virus particles (NL 730 nm) were observed; this virus being apparently a member of the Potyvirus group, resembled as far as its symptoms in papaya are concerned, the papaya ringspot or the distortion ringspot. In Cambodia some young rubber trees showed malformed leaves (esp. edges and veins) with yellow discolorations along the veins. Such leaves contained elongated virus-like particles (rigid or slightly flexible) of various length (60 to 880 nm), so that their normal length (NL) could not be established precisely. Particles 120 to 150 nm long occurred very frequently.     

Trafficking and localization studies of recombinant alpha1, 3- fucosyltransferase VI stably expressed in CHO cells

Peripheral alpha1,3-fucosylation of glycans occurs by the action of either one of five different alpha1,3-fucosyltransferases (Fuc-Ts) cloned to date. Fuc-TVI is one of the alpha1,3-fucosyltransferases which is capable to synthesize selectin ligands. The major alpha1, 3- fucosyltransferase activity in human plasma is encoded by the gene for fucosyltransferase VI, which presumably originates from liver cells. While the sequence, chromosomal localization, and kinetic properties of Fuc-TVI are known, immunocytochemical localization and trafficking studies have been impossible because of the lack of specific antibodies. Here we report on the development and characterization of a peptide-specific polyclonal antiserum monospecific to Fuc-TVI and an antiserum to purified soluble recombinant Fuc-TVI crossreactive with Fuc-TIII and Fuc-TV. Both antisera were applied for immunodetection in stably transfected CHO cells expressing the full-length form of this enzyme (CHO clone 61/11). Fuc-TVI was found to be a resident protein of the Golgi apparatus. In addition, more than 30% of cell-associated and released enzyme activity was found in the medium. Maturation and release of Fuc-TVI was analyzed in metabolically labeled CHO 61/11 cells followed by immunoprecipitation. Fuc-TVI occurred in two forms of 47 kDa and 43 kDa bands, while the secreted form was detected as a 43 kDa. These two different intracellular forms arose by posttranslational modification, as shown by pulse-chase experiments. Fuc-TVI was released to the supernatant by proteolytic cleavage as a partially endo-H resistant glycoform.      

Effects of pH control with phthalate buffers on hot-water extraction of hemicelluloses from spruce wood

Ground spruce wood was extracted with water at 170 °C at four different pH levels (3.8, 4.0, 4.2 and 4.4) achieved by using phthalate buffers. Static batch extractions were carried out in an accelerated solvent extractor (ASE-300). The extracted non-cellulosic carbohydrates, predominantly galactoglucomannans (GGMs), were characterised mainly by sugar unit analysis and molar mass determination. Compared to extraction with plain water, extractions with phthalate buffer solutions gave similar yields of non-cellulosic carbohydrates, but gave up to 70% less monosaccharides, and consequently higher molar masses of extracted GGMs. Moreover, at these pH levels, the hydrolysis of acetyl groups were decreased by 40% compared to extraction with plain water, thus maintaining the water solubility of GGMs. It is concluded that hot water extraction of hemicelluloses in high-molar-mass form (average Mw about 10 kDa) from wood in good yields (8% of wood) demands appropriate control of pH, to a level of about 4.