Cloning, nucleotide sequence, and expression of the flavodoxin gene from Desulfovibrio vulgaris (Hildenborough)

The gene coding for the flavodoxin protein from Desulfovibrio vulgaris (Hildenborough) has been identified, cloned, and sequenced. DNA fragments containing the flavodoxin gene were identified by hybridization of a mixed synthetic heptadecanucleotide probe to Southern blots of SalI-digested genomic DNA. The nucleotide sequences of the probe were derived from the published protein primary structure (Dubourdieu, M., LeGall, J., and Fox, J. L. (1973) Biochem. Biophys. Res. Commun. 52, 1418-1425). The same oligonucleotide probe was used to screen libraries (in pUC19) containing size-selected SalI fragments. One recombinant, carrying a 1.6-kilobase (kb) insert which strongly hybridizes to the probe, was found to contain a nucleotide sequence which codes for the first 104 residues of the amino-terminal portion of the flavodoxin protein sequence but lacked the remainder of the gene. Therefore, a PstI restriction fragment from this clone was used as a probe to isolate the entire gene from a partial Sau3AI library in Charon 35. Of the plaques which continued to hybridize strongly to this probe through repeated screenings, one recombinant, containing a 16-kb insert, was further characterized. The entire flavodoxin gene was localized within a 1.4-kb XhoI-SacI fragment of this clone. The complete nucleotide sequence of the structural gene for the flavodoxin protein from Desulfovibrio vulgaris and flanking sequences which may include promoter and regulatory sequences are reported here. The cloned flavodoxin gene was placed behind the hybrid tac promoter for overexpression of the protein in Escherichia coli. Modification to the 5'-end of the gene, including substitutions at the second codon, were required to obtain high levels of expression. The expressed recombinant flavodoxin protein is isolated from E. coli cells as the holoprotein with physical and spectral properties similar to the protein isolated from D. vulgaris. To our knowledge, this is the first example of the expression of a foreign flavodoxin gene in E. coli using recombinant DNA methods.     

Glycosylation sites and site-specific glycosylation in human Tamm- Horsfall glycoprotein

The N-glycosylation sites of human Tamm-Horsfall glycoprotein from one healthy male donor have been characterized, based on an approach using endoproteinase Glu-C (V-8 protease, Staphylococcus aureus ) digestion and a combination of chromatographic techniques, automated Edman sequencing, and fast atom bombardment mass spectrometry. Seven out of the eight potential N-glycosylation sites, namely, Asn52, Asn56, Asn208, Asn251, Asn298, Asn372, and Asn489, turned out to be glycosylated, and the potential glycosylation site at Asn14, being close to the N-terminus, is not used. The carbohydrate microheterogeneity on three of the glycosylation sites was studied in more detail by high-pH anion-exchange chromatographic profiling and 500 MHz1H-NMR spectroscopy. Glycosylation site Asn489 contains mainly di- and tri-charged oligosaccharides which comprise, among others, the GalNAc4 S (beta1-4)GlcNAc terminal sequence. Only glycosylation site Asn251 bears oligomannose-type carbohydrate chains ranging from Man5GlcNAc2to Man8GlcNAc2, in addition to a small amount of complex- type structures. Profiling of the carbohydrate moieties of Asn208 indicates a large heterogeneity, similar to that established for native human Tamm-Horsfall glycoprotein, namely, multiply charged complex-type carbohydrate structures, terminated by sulfate groups, sialic acid residues, and/or the Sda-determinant.      

The elytro-femoral stridulatory apparatus in Curculionidae (Coleoptera), with notes on the acoustic behaviour of Arniticus hylobioides (Boheman 1843) and Erodiscus proximus (Viana 1959), and thanatosis display in the latter species

The elytro-femoral stridulatory apparatus of two species of Curculionidae is described and figured: Erodiscus proximus (Curculioninae: Erodiscini) and Arniticus hylobioides (Molytinae: Hylobiini). Notes on sound generation, acoustical behaviour and phylogenetic implications are given. Thanatosis display is reported for the first time in Erodiscus proximus.     

Summer raids of Arocatus melanocephalus (Heteroptera,Lygaeidae) in urban buildings in Northern Italy: Is climate change to blame?

Starting in 1999, repeated massive intrusions of the bug Arocatus melanocephalus inside urban buildings have been reported every summer in different locations in Northern Italy.

This investigation, performed in the town of Modena, where the problem was particularly intense, by means of a survey and meteorological data series, showed a significant positive correlation between the intensity of insect outbreaks and the daily mean temperature increase.

These findings suggest that current climatic warming might have affected the behaviour and/or population dynamics of A. melanocephalus and that building intrusions might represent an attempt to escape exceptionally high summer temperatures.     

Biomechanical spinal growth modulation and progressive adolescent scoliosis – a test of the 'vicious cycle' pathogenetic hypothesis: Summary of an electronic focus group debate of the IBSE

There is no generally accepted scientific theory for the causes of adolescent idiopathic scoliosis (AIS). As part of its mission to widen understanding of scoliosis etiology, the International Federated Body on Scoliosis Etiology (IBSE) introduced the electronic focus group (EFG) as a means of increasing debate on knowledge of important topics. This has been designated as an on-line Delphi discussion. The text for this debate was written by Dr Ian A Stokes. It evaluates the hypothesis that in progressive scoliosis vertebral body wedging during adolescent growth results from asymmetric muscular loading in a "vicious cycle" (vicious cycle hypothesis of pathogenesis) by affecting vertebral body growth plates (endplate physes). A frontal plane mathematical simulation tested whether the calculated loading asymmetry created by muscles in a scoliotic spine could explain the observed rate of scoliosis increase by measuring the vertebral growth modulation by altered compression. The model deals only with vertebral (not disc) wedging. It assumes that a pre-existing scoliosis curve initiates the mechanically-modulated alteration of vertebral body growth that in turn causes worsening of the scoliosis, while everything else is anatomically and physiologically 'normal' The results provide quantitative data consistent with the vicious cycle hypothesis. Dr Stokes' biomechanical research engenders controversy. A new speculative concept is proposed of vertebral symphyseal dysplasia with implications for Dr Stokes' research and the etiology of AIS. What is not controversial is the need to test this hypothesis using additional factors in his current model and in three-dimensional quantitative models that incorporate intervertebral discs and simulate thoracic as well as lumbar scoliosis. The growth modulation process in the vertebral body can be viewed as one type of the biologic phenomenon of mechanotransduction. In certain connective tissues this involves the effects of mechanical strain on chondrocytic metabolism a possible target for novel therapeutic intervention.     

Quasi-adaptive response to alkylating agents and Ada-protein functions in <Emphasis Type="Italic">Escherichia coli</Emphasis>

In the exponentially growing E. coli cells we have described in 2005 a new fundamental genetic phenomenon, namely quasi-adaptive response to alkylating compounds, “quasi-Ada”. Phenotypic expression of “quasi-Ada” is similar to the true Ada response, however in contrast it develops in the course of pretreatment of the cells by sublethal dose of non-alkylating agent, an NO-containing dinitrosyl iron complex with glutathione (DNIC_glu). To reveal the mechanisms of quasi-adaptation and its association with the function of the regulatory protein Ada here we used a unique property of dual gene expression regulation of aidB1 gene, a part of Ada-regulon, namely its relative independence from Ada protein in anaerobic conditions. Based on the results of aidB1 gene expression analysis an EPR spectra of E.coli MV2176 cells (aidB1::lacZ) in aerobic and anaerobic conditions after the corresponding treatments we concluded that the function and the spatial structure of ^meAda and [(Cys^?)₂Fe⁺(NO⁺)₂]Ada are identical and thus the nitrosylated protein represents an Ada regulon genes expression regulator during quasi-adaptation development.     

Cardioprotective efficacy of dinitrosyl iron complex with L-cysteine in rats in vivo

The effect of dinitrosyl iron complex (DNIC) with L-cysteine on the hemodynamic indices and the size of myocardial infarction, which was induced by 40-min regional ischemia and subsequent 60-min reperfusion, have been studied in rats in vivo. Intravenous bolus injection of DNIC (3.1 μmol/kg body weight in 0.5 ml saline) was performed before regional ischemia; the control group was administered the same volume of saline. DNIC administration significantly decreased the mean blood pressure throughout the experiment. DNIC reduced the duration of cardiac arrhythmias to 170 ± 10 s as against 445 ± 30 s in control. The myocardial infraction size significantly decreased in the DNIC group compared to control (38.0 ± 1.4 and 48.0 ± 3.9% of the area at risk, respectively; p < 0.05). A combination of the vasodilatory effect of DNIC with the reduction of the damaging effect of cardiac ischemia and reperfusion encourage the development of hypotensive and antiischemic drugs on this class of NO donors.     

Metabolism of (-)-grandisin from Piper solmsianum in Coleoptera and Lepidoptera species

The biotransformation of the major Piper solmsianum leaf phenylpropanoids, such as the tetrahydrofuran lignan grandisin and derivatives was investigated in the beetle Naupactus bipes as well as in the caterpillars Heraclides hectorides and Quadrus u-lucida. Analysis of fecal material indicated that metabolism occurred mainly through mono- and di-O-demethylation at para positions of 3,4,5-trimethoxyphenyl rings of tetrahydrofuran lignans during digestion by these insects. Additionally, 3-hydroxy-4,5-dimethoxycinnamyl and 3,4,5-trimethoxycinnamyl alcohols were identified in fecal extracts of N. bipes.     

Quasi-adaptive response to alkylating agents in Escherichia coli and Ada-protein functions

In 2005 we have described in exponentially growing E. coli cells a new fundamental genetic phenomenon,--quasi-adaptive response to alkylating compounds (quasi-Ada). Phenotypic expression of quasi-Ada is similar to the true Ada response. However, in contrast to the letter, it develops in the course of pretreatment of the cells by a sublethal dose of nonalkylating agent, an NO-containing dinitrosyl iron complex with glutathione (DNICglu). To reveal the mechanisms of quasi-adaptation and its association with the function of the Ada regulatory protein, here we used a unique property of dual gene expression regulation of aidB1 gene, a part of the Ada-regulon, namely its relative independence from Ada protein in anaerobic conditions. Based on the results of aidB1 gene expression analysis an EPR spectra of E. coli MV2176 cells (aidB1::lacZ) in aerobic and anaerobic conditions after the corresponding treatments, we conclude that the function and the spatial structure of meAda and [(Cys-)2Fe+(NO+)2]Ada are identical and thus the nitrosylated protein represents a regulator of the Ada regulon gene expression during quasi-adaptation development.     

The mechanisms of S-nitrosothiol decomposition catalyzed by iron.

The mechanisms of S-nitrosothiol transformation into paramagnetic dinitrosyl iron complexes (DNICs) with thiol- or non-thiol ligands or mononitrosyl iron complex (MNICs) with N-methyl-D-glucamine dithiocarbamate catalyzed by iron(II) ions under anaerobic conditions were studied by monitoring EPR or optical features of the complexes and S-nitrosothiols. The kinetic investigations demonstrated the appearance of short-living paramagnetic mononitrosyl-iron complex with L-cysteine prior to the formation of stable dinitrosyl-iron complex with cysteine in the solution of iron(II)-citrate complex (50-100 microM), S-nitrosocysteine (400 microM), and L-cysteine (20 mM) in 100 mM Hepes buffer (pH 7.4). The addition of deoxyhemoglobin (100 microM) did not influence the process, which points to a direct interaction between S-nitrosocysteine and iron(II) ions to yield DNIC. The reaction of DNIC-cysteine formation is first- and second-order in iron and S-nitrosocysteine, respectively. The third-order rate constant is (1.0 +/- 0.2) x 10(5) M(-2) s(-1) (estimated from EPR results) or (2.0 +/- 0.1) x 10(4) M(-2) s(-1) (estimated by optical method). A similar process of DNIC-cysteine formation was observed in a solution of iron(II)-citrate complex, L-cysteine, and NO-proline (200 microM) as a NO* donor. The appearance of a less stable dinitrosyl-iron complex with phosphate was detected when solutions of iron(II)-citrate containing 100 mM phosphate buffer (pH 7.4) were mixed with S-nitrosocysteine or NO-proline. The rapid formation of DNIC with phosphate was followed by its decay. When the concentration of L-cysteine in solutions was reduced from 20 to 1 mM, the life-time of the DNIC-cysteine diminished notably; this was caused by consumption of L-cysteine in the process of DNIC-cysteine formation from S-nitrosocysteine and iron. Thus, L-cysteine is consumed. Formation of DNIC with glutathione was also observed in a solution of glutathione (20 mM), S-nitrosoglutathione (400 microM), and iron(II) complex (800 microM) in 100 mM Hepes buffer (pH 7.4), but the rate of formation was about 10 times slower than the formation of the DNIC-cysteine. The rate of MNIC-MGD formation from iron(II)-MGD complexes and S-nitrosocysteine was first-order in both reactants. The second-order rate constant for this reaction, estimated from EPR measurements, was 30 +/- 5 M(-1) s(-1). Rate constants of MNIC-MGD formation from iron(II)-MGD and the more stable S-nitrosoglutathione and S-nitroso-D,L-penicillamine were equal to 3.0 +/- 0.3 and 0.3 +/- 0.05 M(-1) s(-1), respectively. Thus, the concerted mechanism of DNIC and MNIC formation from S-nitrosothiols and iron(II) ions can be suggested to be predominant.