Evolutionary relationship between different subgroups of restriction endonucleases

The type II restriction endonuclease SsoII shows sequence similarity with 10 other restriction endonucleases, among them the type IIE restriction endonuclease EcoRII, which requires binding to an effector site for efficient DNA cleavage, and the type IIF restriction endonuclease NgoMIV, which is active as a homotetramer and cleaves DNA with two recognition sites in a concerted reaction. We show here that SsoII is an orthodox type II enzyme, which is active as a homodimer and does not require activation by binding to an effector site. Nevertheless, it shares with EcoRII and NgoMIV a very similar DNA-binding site and catalytic center as shown here by a mutational analysis, indicative of an evolutionary relationship between these three enzymes. We suggest that a similar relationship exists between other orthodox type II, type IIE, and type IIF restriction endonucleases. This may explain why similarities may be more pronounced between members of different subtypes of restriction enzymes than among the members of a given subtype.     

Delayed striate cortical activation during spatial attention

Recordings of event-related potentials (ERPs) and event-related magnetic fields (ERMFs) were combined with functional magnetic resonance imaging (fMRI) to study visual cortical activity in humans during spatial attention. While subjects attended selectively to stimulus arrays in one visual field, fMRI revealed stimulus-related activations in the contralateral primary visual cortex and in multiple extrastriate areas. ERP and ERMF recordings showed that attention did not affect the initial evoked response at 60-90 ms poststimulus that was localized to primary cortex, but a similarly localized late response at 140-250 ms was enhanced to attended stimuli. These findings provide evidence that the primary visual cortex participates in the selective processing of attended stimuli by means of delayed feedback from higher visual-cortical areas.     

New diacylated 2-hydroxythymol derivatives from Melampodium divaricatum

Melampodium divaricatum is a medicinal plant, which occurs in Central America. In a recent paper we reported the occurrence of acylated 2-hydroxy thymol glycosides as main constituents in this plant. This paper deals with the isolation of two new 2,5-dihydroxythymol ester derivatives. The formerly reported sesquiterpene lactone mikanokryptin was not found in our plant material.     

Branchial mitochondria-rich cells in the dogfish Squalus acanthias

In marine teleost fishes, the gill mitochondria-rich cells (MRCs) are responsible for NaCl elimination; however, in elasmobranch fishes, the specialized rectal gland is considered to be the most important site for salt secretion. The role of the gills in elasmobranch ion regulation, although clearly shown to be secondary, is not well characterized. In the present study, we investigated some morphological properties of the branchial MRCs and the localization, and activity of the important ionoregulatory enzyme Na(+)/K(+)-ATPase, under control conditions and following rectal gland removal (1 month) in the spiny dogfish, Squalus acanthias. A clear correlation can be made between MRC numbers and the levels of Na(+)/K(+)-ATPase activity in crude gill homogenates (r(2)=-0.69). Strong Na(+)/K(+)-ATPase immunoreactivity is also clearly associated with the basolateral membrane of these MRCs. In addition, the dogfish were able to maintain ionic balance after rectal gland removal. These results all suggest a possible role of the dogfish gill in salt secretion. MRCs were, however, unresponsive to rectal gland removal in terms of changes in number, fine structure and Na(+)/K(+)-ATPase activity, as might be expected if they were compensating for the loss of salt secretion by the rectal gland. Thus, the specific role that these MRCs play in ion regulation in the dogfish remains to be determined     

Jasmonate-induced lipid peroxidation in barley leaves initiated by distinct 13-LOX forms of chloroplasts

In addition to a previously characterized 13-lipoxygenase of 100 kDa encoded by LOX2:Hv:1 [V?r?s et al., Eur. J. Biochem. 251 (1998), 36-44], two full-length cDNAs (LOX2:Hv:2, LOX2:Hv:3) were isolated from barley leaves (Hordeum vulgare cv. Salome) and characterized. Both of them encode 13-lipoxygenases with putative target sequences for chloroplast import. Immunogold labeling revealed preferential, if not exclusive, localization of lipoxygenase proteins in the stroma. The ultrastructure of the chloroplast was dramatically altered following methyl jasmonate treatment, indicated by a loss of thylakoid membranes, decreased number of stacks and appearance of numerous osmiophilic globuli. The three 13-lipoxygenases are differentially expressed during treatment with jasmonate, salicylate, glucose or sorbitol. Metabolite profiling of free linolenic acid and free linoleic acid, the substrates of lipoxygenases, in water floated or jasmonate-treated leaves revealed preferential accumulation of linolenic acid. Remarkable amounts of free 9- as well as 13-hydroperoxy linolenic acid were found. In addition, metabolites of these hydroperoxides, such as the hydroxy derivatives and the respective aldehydes, appeared following methyl jasmonate treatment. These findings were substantiated by metabolite profiling of isolated chloroplasts, and subfractions including the envelope, the stroma and the thylakoids, indicating a preferential occurrence of lipoxygenase-derived products in the stroma and in the envelope. These data revealed jasmonate-induced activation of the hydroperoxide lyase and reductase branch within the lipoxygenase pathway and suggest differential activity of the three 13-lipoxygenases under different stress conditions.     

CSN1 N-terminal-dependent activity is required for Arabidopsis development but not for Rub1/Nedd8 deconjugation of cullins: a structure-function study of CSN1 subunit of COP9 signalosome

The COP9 signalosome (CSN) is a multifunctional protein complex essential for arabidopsis development. One of its functions is to promote Rub1/Nedd8 deconjugation from the cullin subunit of the Skp1-cullin-F-box ubiquitin ligase. Little is known about the specific role of its eight subunits in deneddylation or any of the physiological functions of CSN. In the absence of CSN1 (the fus6 mutant), arabidopsis CSN complex cannot assemble, which destabilizes multiple CSN subunits and contributes, together with the loss of CSN1, to the phenotype of fus6. To distinguish CSN1-specific functions, we attempted to rescue the complex formation with deletion or point-mutation forms of CSN1 expressed as transgenes in fus6. We show that the central domain of CSN1 is critical for complex assembly, whereas the C-terminal domain has a supporting role. By expressing the C231 fragment, which contains the structural information but lacks the presumed functional domain located at the N terminus, we have rescued the complex formation and restored the Rub1/Nedd8 deconjugation activity on cullins (fus6/C231). Nonetheless, fus6/C231 exhibits pleiotropic phenotype, including photomorphogenic defects and growth arrest at seedling stage. We conclude that CSN1 N-terminal domain is not required for the Rub1/Nedd8 deconjugation activity of cullins, but contributes to a significant aspect of CSN functions that are essential for plant development.     

Role of phosphatidylinositol 3-kinase in the binding of Bordetella pertussis to human monocytes

Bordetella pertussis, the causative agent of whooping cough, adheres to human monocytes by means of filamentous haemagglutinin (FHA), a bacterial surface protein that is recognized by complement receptor type 3 (CR3, alphaMbeta2 integrin). Previous work has shown that an FHA Arg-Gly-Asp (RGD, residues 1097-1099) site interacts with a complex composed of leucocyte response integrin (LRI, alphavbeta3 integrin) and integrin-associated protein (IAP, CD47) on human monocytes, resulting in enhancement of CR3-mediated bacterial binding. However, the pathway that mediates alphavbeta3-alphaMbeta2 integrin signalling remains to be characterized. Here we describe the involvement of phosphatidylinositol 3-kinase (PI3-K) in this pathway. Wortmannin and LY294002, inhibitors of PI3-K, reduced alphavbeta3/IAP-upregulated, CR3-associated bacterial binding to human monocytes. B. pertussis infection of human monocytes resulted in a marked recruitment of cellular PI3-K to the sites of B. pertussis contact. In contrast, cells infected with an isogenic strain carrying a G1098A mutation at the FHA RGD site did not show any recruitment of PI3-K. We found that ligation of FHA by alphavbeta3/IAP induced RGD-dependent tyrosine phosphorylation of a 60 kDa protein, which associated with IAP and PI3-K in human monocytes. These results suggest that PI3-K and a tyrosine phosphorylated 60 kDa protein may be involved in this biologically important integrin signalling pathway.     

A new non-linear normalization method for reducing variability in DNA microarray experiments

Background  

Microarray data are subject to multiple sources of variation, of which biological sources are of interest whereas most others are only confounding. Recent work has identified systematic sources of variation that are intensity-dependent and non-linear in nature. Systematic sources of variation are not limited to the differing properties of the cyanine dyes Cy5 and Cy3 as observed in cDNA arrays, but are the general case for both oligonucleotide microarray (Affymetrix GeneChips) and cDNA microarray data. Current normalization techniques are most often linear and therefore not capable of fully correcting for these effects.     

Light and life--facts and research perspectives at the Cologne Light Symposium 2002

The international meeting Light, Endocrine Systems and Cancer--Facts and Research Perspectives was convened because recent research suggests that ubiquitous light may have more serious cancer consequences than expected. Beyond the established causal link between (over) exposure to sunlight and skin cancer, many scientists consider a causal relationship between light, endocrine systems and internal cancers as biologically plausible. To identify options to turn biological plausibility of mechanisms into true understanding and to assess the possible public health relevance we chose to bring together leading specialists from clinical, experimental laboratory and epidemiological studies of these issues to stimulate a critical, multi-disciplinary discussion of published and new results. Presentations at the symposium covered the physics of light and evolutionary aspects and provided intriguing information about chronobiology, physiology and patho-physiology of endocrine systems and carcinogenesis. Experimental and epidemiological findings on light and skin cancer, and of very recent investigations of relationships between light and internal cancers such as breast cancer were presented. The meeting concluded with a lively discussion of future research options. The symposium's essence and constructive atmosphere are captured in these proceedings [Neuroendocrinol Lett 2002 Jul;23 Suppl 2:1-104] which contain: (i) original presentation papers [Vladimir Anisimov, David Blask, Roland B?ni, George Brainard, Thomas Erren, Alexander Lerchl, Sidney Perkowitz, Chris Portier, Russel Reiter, Richard Stevens, Günter Vollmer]; (ii) abstracts of oral and poster presentations; (iii) four commentaries [Charles Poole, Chris Portier, Till Roenneberg and Rob Lucas, Vladimir Anisimov and Johnni Hansen] on the presentations at the meeting and possible implications for research and public health. In our view, the biological plausibility of mechanistic links between light and hormones and cancer can serve as a unique basis for syn-disciplinary research and we expect that investigations in this area will become a higher priority research focus.     

The putative somatostatin antagonist,cyclo-(7-aminoheptanoyl-Phe-D-Trp-Lys-Thr[BZL]), may act as potent antiproliferative agonist

The cyclic somatostatin (SST) analogue, cyclo-(7-aminoheptanoyl-Phe-D-Trp-Lys-Thr[BZL]) (cSSTA), has been widely used as somatostatin antagonist. In the human neuroblastoma cell line SH-SY5Y the cyclopeptide acts as a somatostatin receptor agonist. Similar to SST, cSSTA inhibits cell proliferation, activates the protein tyrosine phosphatase SHP-2, and stimulates the activity of mitogen-activated protein kinase. These results suggest that in SH-SY5Y neuroblastoma cells somatostatin receptors may exist which exhibit altered antagonist binding properties.