The role of β-ketoacyl-acyl carrier protein synthase III in the condensation steps of fatty acid biosynthesis in sunflower

The β-ketoacyl-acyl carrier protein synthase III (KAS III; EC 2.3.1.180) is a condensing enzyme catalyzing the initial step of fatty acid biosynthesis using acetyl-CoA as primer. To determine the mechanisms involved in the biosynthesis of fatty acids in sunflower (Helianthus annuus L.) developing seeds, a cDNA coding for HaKAS III (EF514400) was isolated, cloned and sequenced. Its protein sequence is as much as 72% identical to other KAS III-like ones such as those from Perilla frutescens, Jatropha curcas, Ricinus communis or Cuphea hookeriana. Phylogenetic study of the HaKAS III homologous proteins infers its origin from cyanobacterial ancestors. A genomic DNA gel blot analysis revealed that HaKAS III is a single copy gene. Expression levels of this gene, examined by Q-PCR, revealed higher levels in developing seeds storing oil than in leaves, stems, roots or seedling cotyledons. Heterologous expression of HaKAS III in Escherichia coli altered their fatty acid content and composition implying an interaction of HaKAS III with the bacterial FAS complex. Testing purified HaKAS III recombinant protein by adding to a reconstituted E. coli FAS system lacking condensation activity revealed a novel substrate specificity. In contrast to all hitherto characterized plant KAS IIIs, the activities of which are limited to the first cycles of intraplastidial fatty acid biosynthesis yielding C6 chains, HaKAS III participates in at least four cycles resulting in C10 chains.     

Hormonal regulation of gene expression in the “slender” mutant of barley (Hordeum vulgare L.)

The slender mutant of barley resembles a normal barley plant treated with high doses of gibberellic acid (GA₃). Expression of GA₃-regulated and abscisic acid (ABA)-regulated mRNAs was studied in the endosperm and roots of mutant and wild-type (WT) plants.Production of -amylase (EC 3.2.1.1) by WT embryoless half-grains was dependent on the presence of GA₃, and was prevented by ABA. In contrast, -amylase was produced by half-grains of the slender mutant in the absence of added GA₃, although it was still reduced by ABA. The spectrum of -amylase mRNAs in slender embryoless half-grains incubated in the absence of added GA₃ was the same as in WT endosperm half-grains incubated in the presence of GA₃. These results indicate that the endosperm of the slender mutant exhibits similar properties to WT endosperm treated with GA₃.In roots the expression of an ABA-inducible mRNA was similar in slender and WT seedlings either treated with exogenous ABA or exposed to dehydration. This result, and the effect of ABA on -amylase production by the endosperm, indicate that the slender plants retain sensitivity to ABA.Abbreviations ABA abscisic acid - AMV avian myeloblastosis virus - GA gibberellin - GA₁ gibberellin A₁ - GA₃ gibberellic acid - WT wild-type     

Features of the biotechnologically relevant polyamide family “cyanophycins” and their biosynthesis in prokaryotes and eukaryotes

Cyanophycin, inclusions in cyanobacteria discovered by the Italian scientist Borzi in 1887, were characterized as a polyamide consisting of aspartic acid and arginine. Its synthesis in cyanobacteria was analyzed regarding growth conditions, responsible gene product, requirements, polymer structure and properties. Heterologous expression of diverse cyanophycin synthetases (CphA) in Escherichia coli enabled further enzyme characterization. Cyanophycin is a polyamide with variable composition and physiochemical properties dependent on host and cultivation conditions in contrast to the extracellular polyamides poly-γ-glutamic acid and poly-ε-l-lysine. Furthermore, recombinant prokaryotes and transgenic eukaryotes, including plants expressing different cphA genes, were characterized as suitable for production of insoluble cyanophycin regarding higher yields and modified composition for other requirements and applications. In addition, cyanophycin was characterized as a source for the synthesis of polyaspartic acid or N-containing bulk chemicals and dipeptides upon chemical treatment or degradation by cyanophycinases, respectively. Moreover, water-soluble cyanophycin derivatives with altered amino acid composition were isolated from transgenic plants, yeasts and recombinant bacteria. Thereby, the range of dipeptides could be extended by biological processes and by chemical modification, thus increasing the range of applications for cyanophycin and its dipeptides, including agriculture, food supplementations, medical and cosmetic purposes, synthesis of the polyacrylate substitute poly(aspartic acid) and other applications.     

The role of glutamine synthetase in the regulation of nitrogenase activity (“switch off” effect) in Rhodospirillum rubrum

We have studied the changes in the activities of both nitrogenase (switch off) and glutamine synthetase in Rhodospirillum rubrum upon addition of ammonium ions or glutamine to nitrogen fixing cultures. Both activities decrease drastically and return in a parallel manner when added ammonia is metabolized. The decrease in glutamine synthetase activity does not seem to be primarily due to adenylylation of the enzyme. Addition of glutamine to cells starved for nitrogen results in inactivation of glutamine synthetase but nitrogenase is only partially switched off.Abbreviations CeMe₃NBr Cetyltrimethylammonium bromide - Hepes N-2-hydroxyethyl-piperazine-N-2 sulfonic acid - MSO methionine-D,L-sulfoximine - Tea-Dmg triethanol amine-3,3-dimethylglutaric acid     

Single-site mutations on the catalase–peroxidase from Sinorhizobium meliloti: role of the distal Gly and the three amino acids of the putative intrinsic cofactor

KatB is the only catalase–peroxidase identified so far in Sinorhizobium meliloti. It plays a housekeeping role, as it is expressed throughout all the growth phases of the free-living bacterium and also during symbiosis. This paper describes the functional and structural characterization of the KatB mutants Gly303Ser, Trp95Ala, Trp95Phe, Tyr217Leu, Tyr217Phe and Met243Val carried out by optical and electron spin resonance spectroscopy. The aim of this work was to investigate the involvement of these residues in the catalatic and/or peroxidatic reaction and falls in the frame of the open dispute around the factors that influence the balance between catalatic and peroxidatic activity in heme enzymes. The Gly303 residue is not conserved in any other protein of this family, whereas the Trp95, Tyr217 and Met243 residues are thought to form an intrinsic cofactor that is likely to play a role in intramolecular electron transfer. Spectroscopic investigations show that the Gly303Ser mutant is almost similar to the wild-type KatB and should not be involved in substrate binding. Mutations on Trp95, Tyr217 and Met243 clear out the catalatic activity completely, whereas the peroxidatic activity is maintained or even increased with respect to that of the wild-type enzyme. The k _cat values obtained for these mutants suggest that Trp95 and Tyr217 form a huge delocalized system that provides a pathway for electron transfer to the heme. Conversely, Met243 is likely to be placed close to the binding site of the organic molecules and plays a crucial role in substrate docking.     

The role of carbonic anhydrase VI in bitter taste perception: evidence from the Car6?/? mouse model

Background

Carbonic anhydrase VI (CA VI) is a secretory isozyme of the α-CA gene family. It is highly expressed in the salivary and mammary glands and secreted into saliva and milk. Although CA VI was first described as a gustatory protein, its exact functional roles have remained enigmatic. Interestingly, polymorphism of the CA6 gene was recently linked to bitter taste perception in humans. In this study, we compared the preference of Car6^−/− and wild-type mice for different taste modalities in an IntelliCage monitoring environment. Morphologies of taste buds, tongue papillae, and von Ebner’s glands were evaluated by light microscopy. Cell proliferation and rate of apoptosis in tongue specimens were examined by Ki67 immunostaining and fluorescent DNA fragmentation staining, respectively.

Results

The behavioral follow up of the mice in an IntelliCage system revealed that Car6^−/− mice preferred 3 μM quinine (bitter) solution, whereas wild type mice preferred water. When the quinine concentration increased, both groups preferentially selected water. Histological analysis, Ki67 immunostaining and detection of apoptosis did not reveal any significant changes between tongue specimens of the knockout and wild type mice.

Conclusions

Our knockout mouse model confirms that CA VI is involved in bitter taste perception. CA VI may be one of the factors which contribute to avoidance of bitter, potentially harmful, substances.     

The role of the NTPDase enzyme family in parasites: what do we know, and where to from here?

Nucleoside triphosphate diphosphohydrolases (NTPDases, GDA1_CD39 protein superfamily) play a diverse range of roles in a number of eukaryotic organisms. In humans NTPDases function in regulating the inflammatory and immune responses, control of vascular haemostasis and purine salvage. In yeast NTPDases are thought to function primarily in the Golgi, crucially involved in nucleotide sugar transport into the Golgi apparatus and subsequent protein glycosylation. Although rare in bacteria, in Legionella pneumophila secreted NTPDases function as virulence factors. In the last 2 decades it has become clear that a large number of parasites encode putative NTPDases, and the functions of a number of these have been investigated. In this review, the available evidence for NTPDases in parasites and the role of these NTPDases is summarized and discussed. Furthermore, the processes by which NTPDases could function in pathogenesis, purine salvage, thromboregulation, inflammation and glycoconjugate formation are considered, and the data supporting such putative roles reviewed. Potential future research directions to further clarify the role and importance of NTPDases in parasites are proposed. An attempt is also made to clarify the nomenclature used in the parasite field for the GDA1_CD39 protein superfamily, and a uniform system suggested.     

Does family language matter? The role of foreign language use and family social capital in the educational achievement of immigrant students in Germany

The role of ethnic resources in the educational success of immigrants is highly disputed. Combining arguments of segmented assimilation and Coleman's concept of family social capital, this study investigates whether speaking one's language of origin at home relates to achievement by facilitating the mobilization of resources or their transmission from parents to children. Mediating and moderating mechanisms are disentangled and empirically questioned in regression models that predict the mathematical competences of immigrant students from Turkey, Poland and the former Soviet Union. Based on data from the German National Educational Panel Study, the results contradict the assumption that foreign language use will contribute to education through mechanisms of social capital. Regardless of parental human capital or the group of origin, the language used at home does not affect the students’ learning when German language proficiency is accounted for.     

Peroxidase from Catharanthus roseus (L.) G. Don and the biosynthesis of α-3′,4′-anhydrovinblastine: a specific role for a multifunctional enzyme

Summary. We have characterized a basic peroxidase with -3,4-anhydrovinblastine (AVLB) synthase activity, which was purified from Catharanthus roseus leaves. This enzyme was the single peroxidase isoenzyme detected in C. roseus leaves, and the single AVLB synthase activity detected in C. roseus extracts. It was observed that the monomeric substrates of AVLB, vindoline and catharanthine, are both suitable electron donors for the oxidizing intermediates of the basic peroxidase, compounds I and II. Results also showed that the reaction proceeds by a radical-propagated mechanism. Substrate specificity studies of the enzyme revealed that it was also able to oxidize several common peroxidase substrates, indicating a broad range of substrate specificity that is characteristic of class III plant peroxidases. Cytochemical studies showed that the enzyme is localized in C. roseus mesophyll vacuoles, in individual spots at the inner surface of the tonoplast. This particular location suggests a meaningful spatial organization that led to the proposal of a metabolic channeling model for the peroxidase-mediated synthesis of AVLB. The importance of this type of mechanism in the regulation of peroxidase isoenzyme functions in vivo is discussed. In view of the results obtained it is concluded that the basic peroxidase present in C. roseus leaves fulfills all the requirements to be considered as an AVLB synthase, and it is proposed that this specific function of this multifunctional enzyme is determined by metabolic channeling resulting from specific protein–protein interactions.Correspondence and reprints: Institute for Molecular and Cell Biology, University of Porto, rua Campo Alegre 823, 4150-180 Porto, Portugal.Received March 15, 2002; accepted February 4, 2003; published online August 26, 2003     

The Fine Structural Observation on the Development of Embryo and Endosperm in the Intergene- rfc Crosses of Fadish ♀ (Raphanus sativus L.) Cabbageo ♂ (Brassica oleracea L.)

The present report deals with the structural abnormalities and abortion of the endosperm, and the related abnormal development of the embryos in the intergeneric crosses of radish (Raphanus sativus L.) ♀ with cabbage (Brassica oleracea L.) ♂. The weak development of ER, the occurence of starch grains in chloroplasts, the curious distribution of chloroplasts around the nuclei, and the earlier formation of cellular endosperm are some primary structure abnormalities of endosperm and they may indicate the poor development, low metabolic activities, and precocious growth in the hybrid endosperm. The endosperm abortion, which starts from chalazal end endosperm, comprises the damage of both structure and function of ER membrane, and the disintegration of nuclei and organelles, or even death. The hybrid embryos degenerated very early in 2-cell stage or even zygote stage in case the endosperm Aborted early. In case of endosperm late-aborted, the hybrid embryos grew for quite a long period of time but become slow down as the endosperm showed abnormal development and started to abort. As the results, the embryos were smaller, the embryo propers and the suspensors showed no growth in step with one another, and the embryo cells appeared structural abnormalities, and finally degenerated and aborted.     

Trace metals in the common mussel,Mytilus edulis (L.), and in the alga Fucus vesiculosus (L.) from the region of the sound (Öresund)

Concentrations of zinc, cadmium, lead and iron were determined in whole soft parts of mussels, Mytilus edulis (L.), and in growing tips of the alga Fucus vesiculosus (L.) from a total of 20 locations in the area of the Sound between Sweden and Denmark. Pollution profiles produced for each metal were similar in any one of the species studied but the profiles for metals in M. edulis were distinct from those for metals in F. vesiculosus. Results for mussels agreed broadly with those reported previously for this region, indicating a northward decrease in available metal concentrations. By contrast, results for algae showed few differences in metal availability throughout the study area, although some local effects were evident.The differences in the pollution profiles exhibited by these two organisms depend on their response to different portions of the total trace metal load of ambient waters. Results from the alga agree well with available data on the concentrations of trace metals in solution in waters of the Sound. The alga thus appears to be responding only to metals in solution, as suggested by previous authors. By contrast, the major proportion of the total body load of metals in mussels is derived from ingested phytoplankton. The pollution profiles exhibited by mussels may be explained if phytoplankton from the Baltic Sea contain higher concentrations of trace metals than do those from Kattegat. Possible mechanisms for the production of such differences in the metal contents of the two phytoplankton populations, and the ecological implications of these differences, are briefly discussed.