The Electron Transfer Flavoprotein <Emphasis Type="Italic">fixABCX</Emphasis> Gene Products from <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> Show a NifA-Dependent Promoter Regulation

The complete nucleotide sequence of the A. brasilense fixA, fixB, fixC, and fixX genes is reported here. Sequence similarities between the protein sequences deduced from fixABCX genes and many electron transfer flavoproteins (ETFs) have been noted. Comparison of the amino acid sequences of both subunits of ETF with the A. brasilense fixA and fixB gene products exhibits an identity of 30%. The amino acid sequence of the other two genes, fixC and fixX, revealed similarity with the membrane-bound electron transfer flavoprotein ubiquinone oxidoreductase (ETF-QO). Using site-directed mutagenesis, mutations were introduced in the fixA promoter element of the A. brasilense fixABCX operon and chimeric pfixA-lacZ reporter gene fusions were constructed. The activation of the fixA promoter of A. brasilense is dependent upon the presence of the NifA protein being approximately 7 times less active than the A. brasilense nifH promoter. These results indicate that NifA from Klebsiella pneumoniae activates the fix promoter of A. brasilense and provide further evidence in support of the regulatory model of NifA activation in A. brasilense. Although no specific function has been assigned to the fixABCX gene products they are apparently required for symbiotic nitrogen fixation. An electron-transferring capacity in the nitrogen fixation pathway has been suggested for the fix gene products based on sequence homologies to the ETFs and ETF-QO proteins and by the absence of orthologous electron transfer proteins NifJ and NifF in A. brasilense.     

Discovering lactic acid bacteria by genomics

This review summarizes a collection of lactic acid bacteria that are now undergoing genomic sequencing and analysis. Summaries are presented on twenty different species, with each overview discussing the organisms fundamental and practical significance, nvironmental habitat, and its role in fermentation, bioprocessing, or probiotics. For those projects where genome sequence data were available by March 2002, summaries include a listing of key statistics and interesting genomic features. These efforts will revolutionize our molecular view of Gram–positive bacteria, as up to 15 genomes from the low GC content lactic acid bacteria are expected to be available in the public domain by the end of 2003. Our collective view of the lactic acid bacteria will be fundamentally changed as we rediscover the relationships and capabilities of these organisms through genomics.     

Experimental genetic approaches to addiction

Drugs of abuse are able to elicit compulsive drug-seeking behaviors upon repeated administration, which ultimately leads to the phenomenon of addiction. Evidence indicates that the susceptibility to develop addiction is influenced by sources of reinforcement, variable neuroadaptive mechanisms, and neurochemical changes that together lead to altered homeostasis of the brain reward system. Addiction is hypothesized to be a cycle of progressive dysregulation of the brain reward system that results in the compulsive use and loss of control over drug taking and the initiation of behaviors associated with drug seeking. The view that addiction represents a pathological state of reward provides an approach to identifying the factors that contribute to vulnerability, addiction, and relapse in genetic animal models.     

A marine dinoflagellate, Amphidinium eilatiensis n. sp., from the benthos of a mariculture sedimentation pond in Eilat, Israel

A species of Amphidinium bloomed in a mariculture sedimentation pond that was used to grow bivalves near the Gulf of Eilat, Israel. Its overall length averaged 13 microm, the hypocone was 11 microm, and its width was 8 microm. It has a ventral ridge. The sulcus begins at the longitudinal flagellar pore and does not project forward in the apex toward the transverse flagellar pore and left margin of the cingulum. The sulcus is a very shallow groove that projects variably about a third of the body length toward the antapex. The cingulum is a deep groove as it circles the cell from the left ventral side to the dorsal side and then becomes very shallow on the right ventral side as it arches posterior toward the longitudinal flagellar pore. Using a modified method for studying dinoflagellate chromosomes in the SEM, we observed 31 chromosomes. The plastid is dorsal and peripheral with 6 ventrally projecting peripheral digital lobes that wrap around the sides of the ventral and posterior nucleus. Amphidinium eilatiensis n. sp. is morphologically closest to Amphidinium carterae and Amphidinium rhynchocephalum, but it does not have the obvious thecal plates or polygonal units described for the former species. Instead, it has a series of spicules, bumps, and ridges on its surface. It differs from A. rhynchocephalum by two morphological characters: surface morphology and gross plastid architecture. The amplified fragments of the rDNA from A. eilatiensis n. sp. isolated from 2 separate sedimentation ponds in Eilat include the 3'- end of the SSU rDNA (about 100 nt), the whole ITS region (ITS1 + 5.8S + ITS2) and the 5'-end of the LSU rDNA (about 900 nts). The total length of the sequences ranged from 1,460 nt. (A. eilatiensis isolate #1) to 1,461 nts. (A. eilatiensis isolate #2). The latter sequences are identical, the difference in length being due to three insertions. Amphidinium eilatiensis is genetically more closely related to A. carterae than to A. klebsii, with respectively 2.36% and 6.93% of sequence divergence.     

Spermine cytotoxicity to human colon carcinoma-derived cells (CaCo-2)

Spermine is a constituent of all vertebrate cells. Nevertheless, it exerts toxic effects if it accumulates in cells. Spermine is a natural substrate of the FAD-dependent polyamine oxidase, a constitutive enzyme of many cell types. It has been reported that the toxicity of spermine was enhanced if polyamine oxidase was inhibited. We were interested to examine spermine toxicity to human colon carcinoma-derived CaCo-2 cells because, in contrast to most tumor cell lines, CaCo-2 cells undergo differentiation, which is paralleled by changes in polyamine metabolism. CaCo-2 cells were remarkably resistant to spermine accumulation, presumably because spermine is degraded by polyamine oxidase at a rate sufficient to provide spermidine for the maintenance of growth. Inactivation of polyamine oxidase increased the sensitivity to spermine. A major reason for the enhanced spermine cytotoxicity at low polyamine oxidase activity is presumably the profound depletion of spermidine, and the consequent occupation of spermidine binding sites by spermine. Hydrogen peroxide and the aldehydes 3-aminopropanal and 3-acetamidopropanal, the products of polyamine oxidase-catalyzed splitting of spermine and N ¹-acetylspermine, contribute little to spermine cytotoxicity. Activation of caspase by spermine was insignificant, and the formation of DNA ladders, another indicator of apoptotic cell death, could not be observed. Thus it appears that cell death due to excessive accumulation of spermine in CaCo-2 cells was mainly nonapoptotic. The content of brush border membranes did not change between days 6 and 8 after seeding, and it was not affected by exposure of the cells to spermine. However, the activities of alkaline phosphatase, sucrase, and aminopeptidase in nontreated cells were considerably enhanced during this period, but remained low if cells were exposed to spermine. These changes appear to indicate that differentiation is prevented by intoxication with spermine, although other explanations cannot be excluded. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effects of oxidation on the hydrolysis by cholesterol esterase of sitosteryl esters as compared to a cholesteryl ester

Phytosteryl esters (PE) are used as ingredients in functional food to decrease plasma concentration of low density lipoprotein-cholesterol (LDL-C). Effective impairment of cholesterol absorption by PE suggests that these esters are hydrolyzed by the pancreatic cholesterol esterase (CEase, EC 3.1.1.13) and the liberated sterol may interfere with cholesterol reducing its intestinal absorption. PE-enriched foods are marketed for cooking purposes, and temperature is one of the most important factors leading to the formation of oxidation products. Very little is known about the outcome of PE oxides during the digestive process. A new analytical method based on mass spectrometric detection directly after enzymatic reaction was developed to determine in vitro the activity of CEase on PE and their oxides present in functional food. Using this method, we identified a new inhibitor of CEase: sitosteryl 9,10-dihydroxystearate, which behaves as a non-competitive inhibitor of the hydrolysis of cholesteryl oleate and sitosteryl oleate.     

On the fine structure of the electrocyte of Electrophorus electricus L.

Summary The general ultrastructure of the electrocyte, the basic unit of the electric organs of Electrophorus electricus, is analyzed. Presented herein are detailed observations of the syncytial surface, its fibrillar coat, invaginations of the plasma membrane and synaptic terminals. Using Thiéry's method glycogen granules were identified in the syncytial cytoplasm and inside the synaptic terminals, their size and structure being compatible with the muscular origin of the electric organs, to which the filamentous meshwork found in the cytoplasm may be related. Among the perinuclear-organelles, are dense bodies with crystalline patterns. The mitochondrial matrix contains dense granules, their size and structure varying according to the organ to which they belong and to the fixation method used.This work has been supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Conselho de Ensino para Graduados da UFRJ and Banco Nacional de Desenvolvimento Econômico, FUNTEC-241