Lipoprotein(a): still an enigma?

PURPOSE OF REVIEW: Lipoprotein(a) belongs to the class of the most atherogenic lipoproteins. Despite intensive research - in the last year more than 80 papers have been published on this topic - information is still lacking on the physiological function of lipoprotein(a) and the site of its catabolism. Important advances have been made in the knowledge of these points, which may have some therapeutic implications. RECENT FINDINGS: The association of high lipoprotein(a) values with an increase in risk for coronary events has been documented in further prospective studies. This increased risk may relate to recent findings that apolipoprotein(a) is produced in situ within the vessel wall. In addition, lipoprotein(a) binds and inactivates the tissue factor pathway inhibitor and induces plasminogen activator inhibitor type 2 expression in monocytes. A new antisense oligonucleotide strategy has been proposed which efficiently inhibits apolipoprotein(a) expression in vitro and in vivo. Apolipoprotein(a), however, suppresses angiogenesis and thus may interfere with the infiltration of tumor cells. Finally, the enzymatic activity leading to the formation of apolipoprotein(a) fragments in plasma and their catabolism have been further elucidated. SUMMARY: We are still far away from understanding the pathways involved in lipoprotein(a) catabolism, and the physiological function of this lipoprotein. Recent findings, however, provide new insight into pathomechanisms in patients with increased lipoprotein(a) related to hemostasis, which may serve as a basis for designing new treatment strategies.     

Water relations of six sclerophylls growing near Trieste (Northeastern Italy): has sclerophylly a univocal functional significance?

Abstract

The annual time course of the water relations of six sclerophylls has been studied with the aim of: a) defining strategies adopted for withstanding summer water stress and b) to check whether their common sclerophyllous habitus could represent a case of convergent evolution devoted to a univocal functional role. In particular, Phillyrea latifolia L. showed to behave like a drought tolerant as indicated by deep summer drop in leaf water potential (Ψ₁) to near the full turgor loss point (Ψ_lp) and in leaf relative water content (RWC) as caused by xylem cavitation. Prunus laurocerasus L. and Laurus nobilis L. showed to be drought avoiding water spenders in that their Ψ₁ dropped in summer causing prompt recovery in RWC. This was made possible by low cavitation in their twigs. Ilex aquifolium L. was a typical drought avoiding water saver in that both Ψ₁ and RWC remained at very high levels throughout the year. Quercus ilex L. behaved like a drought avoiding water spender which switched to drought tolerance in the warmest period of the year. This was explained by its high vulnerability to cavitation. The extreme heterogeneity of the six species studied suggested that sclerophylly cannot be interpreted as an anatomical feature leading to a specific and unifying functional role, at least as regards drought resistance.     

Wild-type phosphoribosylpyrophosphate synthase (PRS) from Mycobacterium tuberculosis: a bacterial class II PRS?

The 5-phospho-α-D-ribose 1-diphosphate (PRPP) metabolite plays essential roles in several biosynthetic pathways, including histidine, tryptophan, nucleotides, and, in mycobacteria, cell wall precursors. PRPP is synthesized from α-D-ribose 5-phosphate (R5P) and ATP by the Mycobacterium tuberculosis prsA gene product, phosphoribosylpyrophosphate synthase (MtPRS). Here, we report amplification, cloning, expression and purification of wild-type MtPRS. Glutaraldehyde cross-linking results suggest that MtPRS predominates as a hexamer, presenting varied oligomeric states due to distinct ligand binding. MtPRS activity measurements were carried out by a novel coupled continuous spectrophotometric assay. MtPRS enzyme activity could be detected in the absence of P(i). ADP, GDP and UMP inhibit MtPRS activity. Steady-state kinetics results indicate that MtPRS has broad substrate specificity, being able to accept ATP, GTP, CTP, and UTP as diphosphoryl group donors. Fluorescence spectroscopy data suggest that the enzyme mechanism for purine diphosphoryl donors follows a random order of substrate addition, and for pyrimidine diphosphoryl donors follows an ordered mechanism of substrate addition in which R5P binds first to free enzyme. An ordered mechanism for product dissociation is followed by MtPRS, in which PRPP is the first product to be released followed by the nucleoside monophosphate products to yield free enzyme for the next round of catalysis. The broad specificity for diphosphoryl group donors and detection of enzyme activity in the absence of P(i) would suggest that MtPRS belongs to Class II PRS proteins. On the other hand, the hexameric quaternary structure and allosteric ADP inhibition would place MtPRS in Class I PRSs. Further data are needed to classify MtPRS as belonging to a particular family of PRS proteins. The data here presented should help augment our understanding of MtPRS mode of action. Current efforts are toward experimental structure determination of MtPRS to provide a solid foundation for the rational design of specific inhibitors of this enzyme.     

Does a functional relationship exist between the polymerization and catalytic activity of beef liver glutamate dehydrogenase?

The recent work of Cohen &; Benedek (1976) and Cohen et al. (1975, 1976) on the apparent interdependence of beef liver glutamate dohydrogenase catalytic activity and degree of polymerization is examined in the light of previously published equilibrium and kinetic results. It is shown that some of the hypotheses central to the Cohen &; Benedek (1976) model are in contradiction with existent data. Consideration of all available information leads to the conclusion that effector-induced depolymerization may simply be an incidental side reaction in the events leading to inhibition.     

Characterization of a chalcone synthase (CHS) flower-specific promoter from Lilium orential ��Sorbonne��

The first enzyme in the flavonoid pathway, chalcone synthase, is encoded by a gene (CHS) whose expression is normally under developmental control. In our previous studies, an 896-bp promoter region of a flower-specific CHS gene was isolated from Lilium orential ‘Sorbonne’, and designated as PLoCHS. Here, the PLoCHS promoter was fused to the β-glucuronidase (GUS) gene to characterize its spatial and temporal expression in Petunia hybrida ‘Dreams Midnight’ using an Agrobacterium-mediated leaf disc transformation method. Our results demonstrated that GUS expression was present in flowers, but reduced or absent in the other tissues (leaf and stem) examined. In petals, GUS activity reached its peak at flower developmental stage 4, and decreased at later stages. Deletion analysis indicated that even a 307-bp fragment of the PLoCHS promoter could still direct flower-specific expression. Further deletion of the region from −261 to −72 bp resulted in weak expression in different organs, including flowers, leaves and stems. This evidence combined with prediction of cis-acting elements in the PLoCHS promoter suggests that the TACPyAT box located in this promoter plays a key role in the regulation of organ-specific expression.     

Recruitment Behavior During Foraging in the Neotropical Ant Gnamptogenys moelleri (Formicidae: Ponerinae): Does the Type of Food Matter?

Gnamptogenys moelleri nests in bromeliads and feeds on an array of food items, including dead and live animals, and nectar. Field data in Brazilian forests indicate that G. moelleri hunts solitarily, while retrieving is performed both by solitary workers for small items, or by a group of recruited workers for large items. This flexible foraging strategy was investigated in the laboratory through a series of experiments to assess the context in which recruitment is elicited. Three types of food were used: 50% honey solution, large insect prey, and cluster of small insects. For all food types the first encounter by a scout resulted in increased numbers of ants leaving the nest and finding the food in the arena. After finding liquid food or large prey, the forager returns to the nest and transmits information to nestmates about food location on the substrate. The successful scout repeatedly taps the sting on the ground, and recruited ants collectively retrieve the large insect to the nest. On the other hand, there is no transmission of information to nestmates about the location of small clumped prey, although the returning scout induces nestmates to leave the nest and hunt. Because foraging in G. moelleri is restricted mostly to the nest bromeliad, and small worker size (0.5 cm) precludes capturing large prey solitarily, recruitment behavior widens the spectrum of food items consumed by this ant species. Although recruitment behavior in ponerines has already been reported to vary with the type and size of a food source, this study also shows that the transmission of information about food location depends on the type of food found (large prey or liquid food versus cluster of small prey).     

Learning from Past Attempts: Does Classical Biological Control of Noogoora Burr (Asteraceae: Xanthium occidentale) Have a Promising Future?

Noogoora burr (Asteraceae: Xanthium occidentale) is an annual from the Neotropics that is a serious weed of rangeland and agricultural regions in many parts of the world. It was one of the first weeds to be systematically targeted for biological control and there were two major phases in the Australian program; 1929-1940, and 1953-1975. Past attempts at biological control are reviewed to identify opportunities for renewed efforts, and for its historical value. Only one insect species was released in the first phase, but host-specificity restrictions were loosened during the second phase resulting in the release of two other insect species. None have been effective. A fourth agent, released primarily for parthenium weed, has had limited impact. However, the rust, Puccinia xanthii, was accidentally introduced in 1975. It has resulted in complete control of Noogoora burr in much of eastern Australia, but has had little or no impact in far northern Australia. Past failures, judged through hindsight, can be attributed to a failure to identify the full native range, lack of consideration for pathogens prior to 1975, and poor understanding of plant compensatory mechanisms and other challenges that an agent must overcome to be effective. A renewed effort specifically targeting Noogoora burr in far northern Australia holds considerable promise. It should include surveys for new agents within the native range of the Xanthium complex, and a systematic search for isolates of the P. xanthii rust that are climatically adapted to tropical conditions in Australia.     

β(1–3)Glucan synthase activity ofNeurospora crassa: Kinetic analysis of negative effectors

β(1–3)Glucan synthase activity ofNeurospora crassa was inhibited by a number of compounds. Uridine nucleotides were linear competitive inhibitors of enzyme activity. Hill plots for the competitive inhibitors and for the substrate (UDP-glucose) resulted in straight lines with Hill numbers near unity suggesting a single substrate binding site. Tunicamycin, dolichol, or dolichol phosphate did not affect enzyme activity and a lipid-linked intermediate was not detected. Sorbose, gluconolactone, echinocandin B, and papulacandin B were uncompetitive inhibitors. Mixed inhibitor studies revealed that the binding of one uncompetitive inhibitor blocked completely the binding of each of the other uncompetitive inhibitors.     

Purification of an elicitor-induced glucan synthase (callose synthase) from suspension cultures of French bean (Phaseolus vulgaris L.): purification and immunolocation of a probable Mr-65 000 subunit of the enzyme

Membrane preparations from suspension-cultured cells of French bean (Phaseolus vulgaris L.) contained callose synthase (EC 2.4.1.34) activity which was preserved upon solubilisation. Following elicitor treatment of cell cultures, increased activity could be extracted and this increase was maintained during purification. The enzyme was purified by high-pressure liquid chromatography and active fractions showed a variable association of two polypeptides of relative molecular masses (M_r) 55 000 and 65 000, the latter being in excess. The M_r-65 000 polypeptide was purified to homogeneity and an antibody raised to it. This antibody showed complex effects on callose synthase activity when incubated with membrane and soluble extracts. In comparison with other systems, the M_r-55 000 subunit is likely to represent the catalytic subunit while the M_r-65 000 polypeptide is a possible regulatory subunit. The M_r-65 000 polypeptide was immunolocated in membranes at sites of callose synthesis in the plant, in cell plates, in sieve plates, at the plasma membrane-wall interface of wounded cells and in papillae in infected cells. Received: 18 January 1997 / Accepted: 8 May 1997     

Lyase activity of glycogen synthase: Is an elimination/addition mechanism a possible reaction pathway for retaining glycosyltransferases?

Despite the biological relevance of glycosyltrasferases (GTs) and the many efforts devoted to this subject, the catalytic mechanism through which a subclass of this large family of enzymes, namely those that operate with net retention of the anomeric configuration, has not been fully established. Here, we show that in the absence of an acceptor, an archetypal retaining GT such as Pyrococcus abyssi glycogen synthase (PaGS) reacts with its glucosyl donor substrate, uridine 5'-diphosphoglucose (UDP-Glc), to produce the scission of the covalent bond between the terminal phosphate oxygen of UDP and the sugar ring. X-ray diffraction analysis of the PaGS/UDP-Glc complex shows no electronic density attributable to the UDP moiety, but establishes the presence in the active site of the enzyme of a glucose-like derivative that lacks the exocyclic oxygen attached to the anomeric carbon. Chemical derivatization followed by gas chromatography/mass spectrometry of the isolated glucose-like species allowed us to identify the molecule found in the catalytic site of PaGS as 1,5-anhydro-D-arabino-hex-1-enitol (AA) or its tautomeric form, 1,5-anhydro-D-fructose. These findings are consistent with a stepwise S(N) i-like mechanism as the modus operandi of retaining GTs, a mechanism that involves the discrete existence of an oxocarbenium intermediate. Even in the absence of a glucosyl acceptor, glycogen synthase (GS) promotes the formation of the cationic intermediate, which, by eliminating the proton of the adjacent C2 carbon atom, yields AA. Alternatively, these observations could be interpreted assuming that AA is a true intermediate in the reaction pathway of GS and that this enzyme operates through an elimination/addition mechanism.     

Characterisation of a novel endo-xyloglucanase (XcXGHA) from Xanthomonas that accommodates a xylosyl-substituted glucose at subsite −1

A xyloglucan-specific endo-1,4β-glucanase (XcXGHA) from Xanthomonas citri pv. mangiferaeindicae has been cloned, expressed in Escherichia coli, purified and characterised. The XcXGHA enzyme belongs to CAZy family GH74 and has catalytic site residues conserved with other xyloglucanases in this family. At its optimal reaction conditions, pH 7.0 and 40 °C, the enzyme has a k _cat/K _M value of 2.2?×?10⁷ min^?1 M^?1 on a tamarind seed xyloglucan substrate. XcXGHA is relatively stable within a broad pH range (pH 4–9) and up to 50 °C (t _{1/2, 50 °C} of 74 min). XcXGHA is proven to be xyloglucan-specific, and a glycan microarray study verifies that XcXGHA catalyses cleavage of xyloglucan extracted from both monocot and dicot plant species. The enzyme catalyses hydrolysis of tamarind xyloglucan in a unique way by cleaving XXXG into XX and XG (X is xylosyl-substituted glucose; G is unsubstituted glucose), is able to degrade more complex xyloglucans and notably is able to cleave near more substituted xyloglucan motifs such as L [i.e. α-l-Fucp-(1?→?2)-β-d-Galp-(1?→?2)-α-d-Xylp-(1?→?6)-β-d-Glcp]. LC-MS/MS analysis of product profiles of tamarind xyloglucan which had been catalytically degraded by XcXGHA revealed that XcXGHA has specificity for X in subsite ?1. The 3D model suggests that XcXGHA consists of two seven-bladed β-propeller domains with the catalytic center formed by the interface of these two domains, which is conserved in xyloglucanases in the GH74 family. However, the XcXGHA has two amino acids (D264 and R472) that differ from the conserved residues of other GH74 xyloglucanases. These two amino acids were predicted to be located on the opposite side of the active site pocket, facing each other and forming a closing surface above the active site pocket. These two amino acids may contribute to the unique substrate specificity of the XcXGHA enzyme.     

Degradation pathway(s) of chlorophyll: what has gene cloning revealed?

The mechanism responsible for the degreening of plants and the degradation of chlorophyll was unclear for many years. However, recent studies have identified the colorless intermediates and helped to construct a basic pathway for degradation. After the successive removal of phytol and Mg21 from the chlorophyll molecule by chlorophyllase and 'Mg dechelatase', pheophorbide a is cleaved and reduced to yield a colorless, open tetrapyrrole intermediate. After further modifications, this is finally transported to the vacuole. Cloning the genes for chlorophyllase isozymes and the reductase should help to elucidate the physiological roles of each enzyme at a molecular level.     

Inositol phosphates: Does IP(4) run a protection racket?

The phosphorylation of IP(3) by IP(3) 3-kinase leads to a number of physiological events, most of which are poorly understood. Recent findings about a hitherto unsuspected action of the IP(3) 3-kinase product, IP(4), suggest that the evolution of IP(3) 3-kinase may have even more far-reaching consequences than we thought.     

Does Ethylene Play a Role in the Release of Lateral Buds (Tillers) from Apical Dominance in Oats?

The growth of lateral buds (tillers), which are undergoing release from apical dominance, was measured in upright and gravistimulated intact Avena sativa L. cv. `Victory' (oat) shoots as well as in isolated Avena stem segments treated with kinetin and sucrose. During release, the tiller bud initially shows a slow rate of elongation accompanied by swelling. It is followed by a more rapid rate of elongation. Ethylene (C₂H₄) production in shoot segments containing a tiller bud was found to occur at the onset of tiller swelling during gravistimulation as well as during inflorescence emergence. Exogenous application of indoleacetic acid or C₂H₄ inhibits kinetin-induced tiller bud swelling and elongation. However, stem segments pulsed for 24 hours in C₂H₄ or the C₂H₄ biosynthesis precursor, 1-amino-cyclopropane-1-carboxylic acid (ACC) and then transferred to kinetin and sucrose, showed a significant increase in swelling elongation as compared with segments maintained under the same conditions but without C₂H₄ or ACC in the pulse. Segments pulsed for 24 hours with kinetin and sucrose plus the ACC biosynthesis inhibitor, aminoethoxyvinylglycine, or the C₂H₄ action inhibitor, CO₂, then transferred to kinetin and sucrose medium, showed inhibition of tiller swelling during the pulse and of subsequent elongation. These results indicate that C₂H₄ plays a role in promoting tiller swelling during the onset of tiller release from apical dominance and may act as a modulator hormone in promoting tiller elongation in the presence of cytokinin.