The serotonin transporter (SERT)2 is an integral membrane protein that facilitates transport of serotonin (5-hydroxytryptamine, 5HT) across cellular membranes (1). In addition to peripheral endocrine functions, 5HT is a neurotransmitter in the brain; it is involved in control of several important physiological functions such as mood, appetite, and sexual behavior. Expressed mainly in the membrane of serotonergic neurons, SERT utilizes energetically favorable cotransport of Na ϩ to remove released 5HT from the extracellular space. Human SERT (hSERT) belongs to the solute carrier 6 (SLC6) transporter family along with highly homologous transporters for the neurotransmitters ␥-aminobutyric acid, glycine, dopamine, and norepinephrine (2-4). These transporters are important drug targets for treatment of a wide range of neurological diseases. In particular, hSERT is the molecular target for widely used drugs for treatment of depression and anxiety. Also, psychostimulants such as amphetamine and 3,4-methylenedioxy-N-methylamphetamine ("ecstasy") have hSERT as the molecular target (5-7). The selective serotonin re-uptake inhibitors (SSRIs) are a class of antidepressant and anti-anxiety drugs that function as highly selective competitive inhibitors of hSERT (8). Although SSRIs are highly important for treatment of affective disorders (6), the molecular basis for their function, including location and structure of drug binding pockets, is largely unknown and a matter of debate (9, 10). Such information is important for understanding essential aspects of drug action, ranging from selectivity profile to therapeutic efficacy. Moreover, such information is indispensable for the development of new and improved drugs targeting hSERT. The primary impediment for elucidation of the structural mechanisms of hSERT inhibition is the lack of a three-dimensional structure of the protein. Still, several residues in SERT have been identified mainly by mutagenesis studies that modulate antidepressants potency (11)(12)(13)(14)(15)(16)(17). The use of comparative molecular modeling to create structural models of ligand-hSERT interactions has previously been hampered by the low phylogenetic and functional similarity between hSERT and available template proteins (18 -21). However, high resolution crystal structures of a bacterial homolog to mammalian SLC6 transporters, LeuT (22,23), have proven excellent templates for constructing experimentally validated models of substrate and drug binding pockets in human SLC6 transporters, including the human transporters for dopamine and ␥-aminobutyric acid (24 -32).In this study, we provide an experimentally validated threedimensional model of the binding site in hSERT for the SSRI (S)-citalopram (Lexapro) using mutational analysis of hSERT paired with structure-activity data for (S)-citalopram analogs. LeuT structures are used to create homology models of hSERT, followed by docking of (S)-citalopram. Validation of binding models was performed based on the mutational dataset from 64 hSERT point mutants ...

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Three ␤-galactosidase genes from Bifidobacterium bifidum DSM20215 and one ␤-galactosidase gene from Bifidobacterium infantis DSM20088 were isolated and characterized. The three B. bifidum ␤-galactosidases exhibited a low degree of amino acid sequence similarity to each other and to previously published ␤-galactosidases classified as family 2 glycosyl hydrolases. Likewise, the B. infantis ␤-galactosidase was distantly related to enzymes classified as family 42 glycosyl hydrolases. One of the enzymes from B. bifidum, termed BIF3, is most probably an extracellular enzyme, since it contained a signal sequence which was cleaved off during heterologous expression of the enzyme in Escherichia coli. Other exceptional features of the BIF3 ␤-galactosidase were (i) the monomeric structure of the active enzyme, comprising 1,752 amino acid residues (188 kDa) and (ii) the molecular organization into an N-terminal ␤-galactosidase domain and a C-terminal galactose binding domain. The other two B. bifidum ␤-galactosidases and the enzyme from B. infantis were multimeric, intracellular enzymes with molecular masses similar to typical family 2 and family 42 glycosyl hydrolases, respectively. Despite the differences in size, molecular composition, and amino acid sequence, all four ␤-galactosidases were highly specific for hydrolysis of ␤-D-galactosidic linkages, and all four enzymes were able to transgalactosylate with lactose as a substrate.Since they were first discovered by Tissier (33), the bifidobacteria have been investigated extensively by several scientists (e.g., references 23 and 27). In recent years, bifidobacteria have attracted particular attention due to their promising health-promoting properties, for example, reduction of harmful bacteria and toxic compounds in the intestine, prevention of dental caries, reduction of total cholesterol and lipid in serum, and relief of constipation (2,5,10,17,36,41). Therefore, live probiotic bifidobacteria, which may improve the microbial balance of the human gastrointestinal tract, have been used to supplement dairy products for many years. Another approach to increase the number of beneficial bacteria in the human intestine is to selectively stimulate their growth by supplementing food with ingredients which can only be metabolized by such bacteria. Certain oligosaccharides, the so-called prebiotics, have been shown to exert this growth-stimulating effect on probiotic bacteria, including bifidobacteria.So far, most of the probiotic bacteria and the prebiotic oligosaccharides have been used in combination with dairy products, and since these products often contain large amounts of lactose, much attention has been focused on the enzyme ␤-galactosidase (EC 3.2.1.23), which is involved in the bacterial metabolism of lactose. In addition to normal hydrolysis of the ␤-D-galactoside linkage in lactose, some ␤-D-galactosidase enzymes may catalyze the formation of galactooligosaccharides through transfer of one or more D-galactosyl units onto the D-galactose moiety of lactose. This transgal...

The Lactococcus lactis pfl gene, encoding pyruvate formate-lyase (PFL), has been cloned and characterized. The deduced amino acid sequence of the L. lactis PFL protein showed high similarity to those of other bacterial PFL proteins and included the conserved glycine residue involved in posttranslational activation of PFL. The genetic organization of the chromosomal pfl region in L. lactis showed differences from other characterized pfl loci, with an upstream open reading frame independently transcribed in the same orientation as the pfl gene. The gene coding for PFL-activase (act), normally found downstream of pfl, was not identified in L. lactis. Analysis of pfl expression showed a strong induction under anaerobiosis at the transcriptional level independent of the growth medium used. During growth with galactose, pfl showed the highest levels of expression. Constructed L. lactis pfl strains were unable to produce formate under anaerobic growth. Higher levels of diacetyl and acetoin were produced anaerobically in the constructed Lactococcus lactis subsp. lactis biovar diacetylactis pfl strain.Pyruvate has a key role in the metabolic network of Lactococcus lactis. In this industrially relevant organism, sugars are metabolized predominantly to generate energy and as a carbon source. The catabolism of sugars leads to pyruvate, whose further metabolism determines the nature of the fermentation (see reference 5 for a review). Lactate is the major end product responsible for acidification, while other minor metabolites, mainly diacetyl, are responsible for aroma in fermented milk products. Several genes involved in pyruvate metabolism have been studied for their role in the production of diacetyl (Fig.

The ability to convert D-galactose into D-tagatose was compared among a number of bacterial L-arabinose isomerases ( araA). One of the most efficient enzymes, from the anaerobic thermophilic bacterium Thermoanaerobacter mathranii, was produced heterologously in Escherichia coli and characterised. Amino acid sequence comparisons indicated that this enzyme is only distantly related to the group of previously known araA sequences in which the sequence similarity is evident. The substrate specificity and the Michaelis-Menten constants of the enzyme determined with L-arabinose, D-galactose and D-fucose also indicated that this enzyme is an unusual, versatile L-arabinose isomerase which is able to isomerise structurally related sugars. The enzyme was immobilised and used for production of D-tagatose at 65 degrees C. Starting from a 30% solution of D-galactose, the yield of D-tagatose was 42% and no sugars other than D-tagatose and D-galactose were detected. Direct conversion of lactose to D-tagatose in a single reactor was demonstrated using a thermostable beta-galactosidase together with the thermostable L-arabinose isomerase. The two enzymes were also successfully combined with a commercially available glucose isomerase for conversion of lactose into a sweetening mixture comprising lactose, glucose, galactose, fructose and tagatose.

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A microfluidic component library for building systems driving parallel or serial microfluidic-based assays is presented. The components are a miniaturized eight-channel peristaltic pump, an eight-channel valve, sample-to-waste liquid management, and interconnections. The library of components was tested by constructing various systems supporting perfusion cell culture, automated DNA hybridizations, and in situ hybridizations. The results showed that the MainSTREAM components provided (1) a rapid, robust, and simple method to establish numerous fluidic inputs and outputs to various types of reaction chips; (2) highly parallel pumping and routing/valving capability; (3) methods to interface pumps and chip-to-liquid management systems; (4) means to construct a portable system; (5) reconfigurability/flexibility in system design; (6) means to interface to microscopes; and (7) compatibility with tested biological methods. It was found that LEGO Mindstorms motors, controllers, and software were robust, inexpensive, and an accessible choice as compared with corresponding custom-made actuators. MainSTREAM systems could operate continuously for weeks without leaks, contamination, or system failures. In conclusion, the MainSTREAM components described here meet many of the demands on components for constructing and using microfluidics systems.

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