In this paper, we report on engineering 3-D pulmonary tissue constructs in vitro. Primary isolates of murine embryonic day 18 fetal pulmonary cells (FPC) were comprised of a mixed population of epithelial, mesenchymal, and endothelial cells as assessed by immunohistochemistry and RT-PCR of 2-D cultures. The alveolar type II (AE2) cell phenotype in 2-D and 3-D cultures was confirmed by detection of SpC gene expression and presence of the gene product prosurfactant protein C. Three-dimensional constructs of FPC were generated utilizing Matrigel hydrogel and synthetic polymer scaffolds of poly-lactic-co-glycolic acid (PLGA) and poly-L-lactic-acid (PLLA) fabricated into porous foams and nanofibrous matrices, respectively. Three-dimensional Matrigel constructs contained alveolar forming units (AFU) comprised of cells displaying AE2 cellular ultrastructure while expressing the SpC gene and gene product. The addition of tissue-specific growth factors induced formation of branching, sacculated epithelial structures reminiscent of the distal lung architecture. Importantly, 3-D culture was necessary for inducing expression of the morphogenesis-associated distal epithelial gene fibroblast growth factor receptor 2 (FGFr2). PLGA foams and PLLA nanofiber scaffolds facilitated ingrowth of FPC, as evidenced by histology. However, these matrices did not support the survival of distal lung epithelial cells, despite the presence of tissue-specific growth factors. Our results may provide the first step on the long road toward engineering distal pulmonary tissue for augmenting and/or replacing dysfunctional native lung in diseases, such as neonatal pulmonary hypoplasia.

Numerous microorganisms, including bacteria, yeasts, and molds, are present in cheeses, forming a complex ecosystem. Among these organisms, bacteria are responsible for most of the physicochemical and aromatic transformations that are intrinsic to the cheesemaking process. Identification of the bacteria that constitute the cheese ecosystem is essential for understanding their individual contributions to cheese production. We used temporal temperature gradient gel electrophoresis (TTGE) to identify different bacterial species present in several dairy products, including members of the genera Lactobacillus, Lactococcus, Leuconostoc, Enterococcus, Pediococcus, Streptococcus, and Staphylococcus. The TTGE technique is based on electrophoretic separation of 16S ribosomal DNA (rDNA) fragments by using a temperature gradient. It was optimized to reveal differences in the 16S rDNA V3 regions of bacteria with low-G+C-content genomes. Using multiple control strains, we first set up a species database in which each species (or group of species) was characterized by a specific TTGE fingerprint. TTGE was then applied to controlled dairy ecosystems with defined compositions, including liquid (starter), semisolid (home-made fermented milk), and solid (miniature cheese models) matrices. Finally, the potential of TTGE to describe the bacterial microflora of unknown ecosystems was tested with various commercial dairy products. Subspecies, species, or groups of species of lactic acid bacteria were distinguished in dairy samples. In conclusion, TTGE was shown to distinguish bacterial species in vitro, as well as in both liquid and solid dairy products.

Dental caries is a chronic infectious disease of multifactorial etiology that derives from the interplay among cariogenic bacteria on the dentition, the host diet, and other environmental exposures. Streptococcus mutans proliferates as a biofilm on the tooth surface, where it obtains nutrients and metabolizes fermentable dietary carbohydrates. The accumulation of lactic acid as a by-product of fermentation results in acidification of the plaque biofilm and demineralization of tooth enamel, marking the onset of decay. The ability of S. mutans to respond to environmental stresses presented by salivary flow, acid pH, oxidative stress, and changes in carbohydrate source and availability is essential for its survival and predominance in caries lesions. Importantly, S. mutans has evolved a network of regulators to integrate its cellular response to environmental change. Herein we describe the latest insights into global gene regulation in S. mutans, including mechanisms of signal transduction, carbon catabolite repression, and quorum-sensing. An improved understanding of these regulatory networks can provide a basis for novel therapeutic applications aimed at treating and/or preventing caries.

Fermentative production of optically pure lactic acid has roused interest among researchers in recent years due to its high potential for applications in a wide range of fields. More specifically, the sharp increase in manufacturing of biodegradable polylactic acid (PLA) materials, green alternatives to petroleum-derived plastics, has significantly increased the global interest in lactic acid production. However, higher production costs have hindered the large-scale application of PLA because of the high price of lactic acid. Therefore, reduction of lactic acid production cost through utilization of inexpensive substrates and improvement of lactic acid production and productivity has become an important goal. Various methods have been employed for enhanced lactic acid production, including several bioprocess techniques facilitated by wild-type and/or engineered microbes. In this review, we will discuss lactic acid producers with relation to their fermentation characteristics and metabolism. Inexpensive fermentative substrates, such as dairy products, food and agro-industrial wastes, glycerol, and algal biomass alternatives to costly pure sugars and food crops are introduced. The operational modes and fermentation methods that have been recently reported to improve lactic acid production in terms of concentrations, yields, and productivities are summarized and compared. High cell density fermentation through immobilization and cell-recycling techniques are also addressed. Finally, advances in recovery processes and concluding remarks on the future outlook of lactic acid production are presented.

The promise of human embryonic stem cells (hESCs) to provide an unlimited supply of cells for cell therapy and tissue engineering depends on the availability of a controllable bioprocess for their expansion and differentiation. We describe for the first time the formation of differentiating human embryoid bodies (hEBs) in rotating bioreactors to try and control their agglomeration. The efficacy of the dynamic process compared to static cultivation in Petri dishes was analyzed with respect to the yield of hEB formation and differentiation. Quantitative analyses of hEBs, DNA and protein contents, and viable cell concentration, as measures for culture cellularity and scale-up, revealed 3-fold enhancement in generation of hEBs compared to the static culture. Other metabolic indices such as glucose consumption, lactic acid production, and pH pointed to efficient cell expansion and differentiation in the dynamic cultures. The type of rotating vessel had a significant impact on the process of hEB formation and agglomeration. In the slow turning lateral vessel (STLV), hEBs were smaller in size and no large necrotic centers were seen, even after 1-month cultivation. In the high aspect rotating vessel (HARV), hEB agglomeration was massive. The appearance of representative tissues derived from the three germ layers as well as primitive neuronal tube organization, blood vessel formation, and specific-endocrine secretion indicated that the initial developmental events are not altered in the dynamically formed hEBs. Collectively, our study defines the culture conditions in which control over the aggregation of differentiating hESCs is obtained, thus enabling scaleable cell production for clinical and industrial applications.

In the representative gut bacterium Lactobacillus plantarum, we identified genes encoding the enzymes involved in a saturation metabolism of polyunsaturated fatty acids and revealed in detail the metabolic pathway that generates hydroxy fatty acids, oxo fatty acids, conjugated fatty acids, and partially saturated trans-fatty acids as intermediates. Furthermore, we observed these intermediates, especially hydroxy fatty acids, in host organs. Levels of hydroxy fatty acids were much higher in specific pathogen-free mice than in germ-free mice, indicating that these fatty acids are generated through polyunsaturated fatty acids metabolism of gastrointestinal microorganisms. These findings suggested that lipid metabolism by gastrointestinal microbes affects the health of the host by modifying fatty acid composition.

The effects of poly(D,L-lactic acid) macroporous guidance scaffolds (foams) with or without brain-derived neurotrophic factor (BDNF) on tissue sparing, neuronal survival, axonal regeneration, and behavioral improvements of the hindlimbs following implantation in the transected adult rat thoracic spinal cord were studied. The foams were embedded in fibrin glue containing acidic-fibroblast growth factor. One group of animals received fibrin glue with acidic-fibroblast growth factor only. The foams were prepared by a thermally induced polymer-solvent phase separation process and contained longitudinally oriented macropores connected to each other by a network of micropores. Both foams and fibrin only resulted in a similar gliotic and inflammatory response in the cord-implant interfaces. With BDNF foam, up to 20% more NeuN-positive cells in the spinal nervous tissue close to the rostral but not caudal spinal cord-implant interface survived than with control foam or fibrin only at 4 and 8 weeks after implantation. Semithin plastic sections and electron microcopy revealed that cells and axons more rapidly invaded BDNF foam than control foam. Also, BDNF foam contained almost twice as many blood vessels than control foam at 8 weeks after implantation. Tissue sparing was similar in all three implantation paradigms; approximately 42% of tissue was spared in the rostral cord and approximately 37% in the caudal cord at 8 weeks post grafting. The number of myelinated and unmyelinated axons was low and not different between the two types of foams. Many more axons were found in the fibrin only graft. Serotonergic axons were not found in any of the implants and none of the axons regenerated into the caudal spinal cord. The behavioral improvements in the hindlimbs were similar in all groups. These findings indicated that foam is well tolerated within the injured spinal cord and that the addition of BDNF promotes cell survival and angiogenesis. However, the overall axonal regeneration response is low. Future research should explore the use of poly(D,L-lactic acid) foams, with or without axonal growth-promoting factors, seeded with Schwann cells to enhance the axonal regeneration and myelination response.

Pain stimulates some behaviors (e.g., withdrawal responses) but depresses many other behaviors (e.g., feeding). Pain-stimulated behaviors are widely used in preclinical research on pain and analgesia, but human and veterinary medicine often rely on measures of functional impairment and pain-depressed behavior to diagnose pain or assess analgesic efficacy. In view of the clinical utility of measures of pain-depressed behaviors, our laboratory has focused on the development of methods for preclinical assays of pain-depressed behavior in rodents. The present study compared the effects of a chemical noxious stimulus (IP lactic acid injections) and an opioid analgesic (morphine) administered alone or in combination on the stretching response (a pain-stimulated behavior) and intracranial self-stimulation (ICSS; a behavior that may be depressed by pain) in rats. In the ICSS procedure, rats implanted with electrodes in the lateral hypothalamus responded to electrical stimulation across a range of current frequencies to permit rapid determination of frequency-rate curves and evaluation of curve shifts following treatment. Lactic acid alone produced a concentration-dependent stimulation of stretching and depression of ICSS, expressed as rightward shifts in ICSS frequency-rate curves. Morphine had little effect alone, but it produced a dose-dependent blockade of both acid-stimulated stretching and acid-depressed ICSS. Both lactic acid and morphine were equipotent in the stretching and ICSS procedures. These results suggest that ICSS may be useful as a behavioral baseline for studies of pain-depressed behavior.

The hypoxia-inducible factor 1 (HIF-1), in addition to genetic and epigenetic changes, is largely responsible for alterations in cell metabolism in hypoxic tumor cells. This transcription factor not only favors cell proliferation through the metabolic shift from oxidative phosphorylation to glycolysis and lactic acid production but also stimulates nutrient supply by mediating adaptive survival mechanisms. In this study we showed that glycogen synthesis is enhanced in non-cancer and cancer cells when exposed to hypoxia, resulting in a large increase in glycogen stores. Furthermore, we demonstrated that the mRNA and protein levels of the first enzyme of glycogenesis, phosphoglucomutase1 (PGM1), were increased in hypoxia. We showed that induction of glycogen storage as well as PGM1 expression were dependent on HIF-1 and HIF-2. We established that hypoxia-induced glycogen stores are rapidly mobilized in cells that are starved of glucose. Glycogenolysis allows these "hypoxia-preconditioned" cells to confront and survive glucose deprivation. In contrast normoxic control cells exhibit a high rate of cell death following glucose removal. These findings point to the important role of hypoxia and HIF in inducing mechanisms of rapid adaptation and survival in response to a decrease in oxygen tension. We propose that a decrease in pO(2) acts as an "alarm" that prepares the cells to face subsequent nutrient depletion and to survive.

The fabrication and surface modification of a porous cell scaffold are very important in tissue engineering. Of most concern are high-density cell seeding, nutrient and oxygen supply, and cell affinity. In the present study, poly(L-lactic acid) and poly(L-lactic-co-glycolic acid) (70/30) cell scaffolds with different pore structures were fabricated. An improved method based on Archimedes' Principle for measuring the porosity of scaffolds, using a density bottle, was developed. Anhydrous ammonia plasma treatment was used to modify surface properties to improve the cell affinity of the scaffolds. The results show that hydrophilicity and surface energy were improved. The polar N-containing groups and positive charged groups also were incorporated into the sample surface. A low-temperature treatment was used to maintain the plasma-modified surface properties effectively. It would do help to the further application of plasma treatment technique. Cell culture results showed that pores smaller than 160 microm are suitable for human skin fibroblast cell growth. Cell seeding efficiency was maintained at above 99%, which is better than the efficiency achieved with the common method of prewetting by ethanol. The plasma-treatment method also helped to resolve the problem of cell loss during cell seeding, and the negative effects of the ethanol trace on cell culture were avoided. The results suggest that anhydrous ammonia plasma treatment enhances the cell affinity of porous scaffolds. Mass transport issues also have been considered.

Genetic lesions responsible for amino acid requirements in several species of multiple auxotrophic lactobacilli were investigated. Systematic attempts were made to isolate mutants that could grow in the absence of each of the amino acids required by the parental strains of Lactobacillus plantarum, L. casei, L. helveticus, and L. acidophilus. After treatment with appropriate mutagens, such mutants could be obtained with respect to many but not all required amino acids. Successful isolation of mutants for a given amino acid means that a minor genetic lesion reparable by single-step mutations affects its biosynthesis; a failure to isolate mutants suggests the involvement of more extensive lesions. Analysis of these results as well as the specific requirements exhibited by the parental strains revealed certain regularities; some of the biosynthetic pathways for individual amino acids were virtually unaffected by more extensive lesions in at least species tested, whereas others were affected by more extensive lesions in at least some species. Both the number and the kind of pathways affected by extensive lesions differed appreciably among different species. Furthermore, the growth response of the parental strains to some putative amino acid precursors revealed a clear correlation between the extent of genetic lesions and the occurrence and location of a genetic block(s) for a given pathway. These findings are discussed in relation to the phylogeny, ecology, and evolution of lactic acid bacteria.

Bacteriophages of lactic acid bacteria are a threat to industrial milk fermentation. Owing to their economical importance, dairy phages became the most thoroughly sequenced phage group in the database. Comparative genomics identified related cos-site and pac-site phages, respectively, in lactococci, lactic streptococci and lactobacilli. Each group was represented with closely related temperate and virulent phages. Over the structural genes their gene maps resembled that of lambdoid coliphages, suggesting distant evolutionary relationships. Despite a lack of sequence similarity, a number of biochemical characteristics of these dairy phages are lambda-like (genetic switch, DNA packaging, head and tail morphogenesis, and integration, but not excision). These dairy phages thus provide interesting variations to the phage lambda paradigm. The structural gene cluster of Lactococcus phage r1t resembled that of phages from mycobacteria. Virulent lactococcal phages with prolate heads (c2-like genus of Siphoviridae), in contrast, have no known counterparts in other bacterial genera.

The proteolytic system of lactic acid bacteria is of eminent importance for the rapid growth of these organisms in protein-rich media. The combined action of proteinases and peptidases provides the cell with small peptides and essential amino acids. The amino acids and peptides thus liberated have to be translocated across the cytoplasmic membrane. To that purpose, the cell contains specific transport proteins. The internalized peptides are further degraded to amino acids by intracellular peptidases. The world-wide economic importance of the lactic acid bacteria and their proteolytic system has led to an intensive research effort in this area and a considerable amount of biochemical data has been collected during the last two decades. Since the development of systems to genetically manipulate lactic acid bacteria, data on the genetics of enzymes and processes involved in proteolysis are rapidly being generated. In this review an overview of the latest genetic data on the proteolytic system of lactic acid bacteria will be presented. As most of the work in this field has been done with lactococci, the emphasis will, inevitably, be on this group of organisms. Where possible, links will be made with other species of lactic acid bacteria.

An understanding of how the heme-deficient gram-positive bacterium Streptococcus pyogenes establishes infections in O(2)-rich environments requires careful analysis of the gene products important in aerobic metabolism. NADH oxidase (NOXase) is a unique flavoprotein of S. pyogenes and other lactic acid bacteria which directly catalyzes the four-electron reduction of O(2) to H(2)O. To elucidate a putative role for this enzyme in aerobic metabolism, NOXase-deficient mutants were constructed by insertional inactivation of the gene that encodes NOXase. Characterization of the resulting mutants revealed that growth in rich medium under low-O(2) conditions was indistinguishable from that of the wild type. However, the mutants were unable to grow under high-O(2) conditions and demonstrated enhanced sensitivity to the superoxide-generating agent paraquat. Mutants cultured in liquid medium under conditions of carbohydrate limitation and high O(2) tension were characterized by an extended lag phase, a reduction in growth, and a greater accumulation of H(2)O(2) in the growth medium compared to the wild-type strain. All of these mutant phenotypes could be overcome by the addition of glucose. Either the addition of catalase to the culture medium of the mutants or the introduction of a heterologous NADH peroxidase into the mutants eliminated the accumulation of H(2)O(2) and rescued the growth defect of the mutants under high-O(2) conditions in carbohydrate-limited liquid medium. Taken together, these data show that NOXase is important for aerobic metabolism and essential in environments high in O(2) with carbohydrate limitation.

The mechanism of metabolic energy production by malolactic fermentation in Lactococcus lactis has been investigated. In the presence of L-malate, a proton motive force composed of a membrane potential and pH gradient is generated which has about the same magnitude as the proton motive force generated by the metabolism of a glycolytic substrate. Malolactic fermentation results in the synthesis of ATP which is inhibited by the ionophore nigericin and the F0F1-ATPase inhibitor N,N-dicyclohexylcarbodiimide. Since substrate-level phosphorylation does not occur during malolactic fermentation, the generation of metabolic energy must originate from the uptake of L-malate and/or excretion of L-lactate. The initiation of malolactic fermentation is stimulated by the presence of L-lactate intracellularly, suggesting that L-malate is exchanged for L-lactate. Direct evidence for heterologous L-malate/L-lactate (and homologous L-malate/L-malate) antiport has been obtained with membrane vesicles of an L. lactis mutant deficient in malolactic enzyme. In membrane vesicles fused with liposomes, L-malate efflux and L-malate/L-lactate antiport are stimulated by a membrane potential (inside negative), indicating that net negative charge is moved to the outside in the efflux and antiport reaction. In membrane vesicles fused with liposomes in which cytochrome c oxidase was incorporated as a proton motive force-generating mechanism, transport of L-malate can be driven by a pH gradient alone, i.e., in the absence of L-lactate as countersubstrate. A membrane potential (inside negative) inhibits uptake of L-malate, indicating that L-malate is transported an an electronegative monoanionic species (or dianionic species together with a proton). The experiments described suggest that the generation of metabolic energy during malolactic fermentation arises from electrogenic malate/lactate antiport and electrogenic malate uptake (in combination with outward diffusion of lactic acid), together with proton consumption as result of decarboxylation of L-malate. The net energy gain would be equivalent to one proton translocated form the inside to the outside per L-malate metabolized.

The two major aims of this study were: (i) to elucidate the underlying release mechanisms from drug-loaded, erodible microparticles based on poly(lactic-co-glycolic acid) (PLGA) showing biphasic drug release behavior: an initial 'burst' effect, followed by a zero order release phase; and (ii) to develop a new, simple mathematical model that allows the quantitative description of the observed in vitro drug release patterns from this type of delivery system. PLGA-based microparticles offer various advantages, such as the possibility to control the resulting drug release rate accurately over prolonged periods of time, easiness of administration (e.g., by stereotaxic injection), good biocompatibility and complete erosion (avoiding the removal of empty remnants). Consequently, the practical importance of these advanced drug delivery systems is remarkably increasing. However, only little knowledge is yet available concerning the processes controlling the release rate of the drug out of these devices. Various chemical and physical phenomena are involved, rendering the identification of the crucial mechanisms and the mathematical description of the resulting drug release kinetics difficult. In the present study, different physicochemical characterization methods (e.g., DSC, SEM, SEC, particle size analysis) were used to monitor the changes occurring within anticancer drug-loaded PLGA microparticles upon exposure to phosphate buffer pH 7.4. Based on these experimental findings, the most important underlying drug release rate controlling mechanisms were identified and a new mathematical model was developed that allows the quantitative description of the resulting release patterns.

A bacteriocin-like inhibitory substance, salivaricin A, was purified from cultures of Streptococcus salivarius 20P3 and was shown by ion spray mass spectrometry to have a molecular mass of 2,315 +/- 1.1 Da. Amino acid composition analysis demonstrated the presence of lanthionine, indicating that salivaricin A may be a member of the lantibiotic class of antibiotic substances. The sequence of eight amino acids at the N terminus of the molecule was determined by Edman degradation, and mixed oligonucleotide probes based on part of this sequence (GSGWIA) were used to detect the salivaricin A structural gene. A 6.2-kb EcoRI fragment of chromosomal DNA from strain 20P3 that hybridized with the probes was cloned, and the hybridizing region was further localized to a 379-bp DraI-AluI fragment. Analysis of the nucleotide sequence of this fragment indicated that salivaricin A is synthesized as a 51-amino-acid prepeptide that is posttranslationally modified and cleaved to give a biologically active 22-residue peptide containing one lanthionine and two beta-methyllanthionine residues. The secondary structure of presalivaricin A was predicted to be similar to that of type A lantibiotics, with a hydrophilic alpha-helical leader sequence and a propeptide region with potential for beta-turn formation and a lack of alpha-helicity. The sequence around the cleavage site of presalivaricin A differed from that of other type A lantibiotics but was similar to that of several bacteriocin-like inhibitory substances produced by lactic acid bacteria.

Ischemia, hypoglycemia, and epilepsy have long been thought to produce similar or identical brain damage. Furthermore, these insults have been assumed to be additive in their damaging effects. These notions have been based on neuropathological observations in the hippocampus and cerebral cortex, and on the tenet that energy failure (ischemia, hypoglycemia) and increased demand for energy (epilepsy) similarly give rise to selective neuronal necrosis. Recently, other bases for considering these three insults identical have grown out of observations that loss of calcium homeostasis is common to all and that an excitotoxic mechanism of selective neuronal necrosis exists in all three conditions. Fundamental differences between ischemia, hypoglycemia, and epilepsy include the underlying neurochemical changes induced, the neuronal revival times, the time course of neuronal death, the distribution of selective neuronal necrosis, and the likely excitotoxins released. Lactic acid accumulation, implicated in damage to the neuropil as well as to neuronal cell bodies, also occurs to different degrees and in different distributions in the three conditions. The degree and distribution of pannecrosis is thus also different in ischemia, hypoglycemia, and epilepsy.

A central debate in the exercise sciences is the cause of the fatigue that develops especially during high intensity exercise of short duration. The most popular theory holds that this form of exercise is limited by a peripherally based, metabolite induced failure of skeletal muscle contractile function, independent of reduced muscle activation by the central nervous system; so-called peripheral fatigue. This theory arose originally from studies undertaken by Nobel Laureate Sir Archibald Vivian Hill and colleagues in Manchester, UK in the 1920s. In turn, their interpretations were crucially influenced by the earlier 1907 findings of Sir Frederick Gowland Hopkins, Nobel Laureate for his discovery of the vitamins, and Walter Morley Fletcher. The original model of Hill and his colleagues proposed that performance during exercise of high intensity was limited by skeletal muscle anaerobiosis that developed as the result of a limiting skeletal muscle blood flow, following the onset of myocardial ischaemia. Such skeletal muscle anaerobiosis ultimately prevented the neutralization of the lactic acid that, Hill believed, initiated muscle contraction. The resulting lactic acid accumulation impaired skeletal muscle relaxation, causing the (involuntary) termination of exercise. The evolutionary progression of this model led to the "catastrophe theory" of Richard Edwards, which posits that exercise terminates when the physiological and biochemical limits of the body are exceeded, causing a catastrophic failure of intracellular homeostasis. This paper addresses six hallmark physiological requirements that must be correct if Hill's cardiovascular/ anaerobic/catastrophic model is the exclusive explanation for the fatigue that develops during maximum exercise to exhaustion. This leads to a review of the evidence supporting other, related "catastrophe" models that have been developed to explain fatigue during exercise of lower intensities and longer durations. It is concluded that there is little published evidence supporting the theory that fatigue occurs only after physiological homeostasis fails according to the prediction of these catastrophe models. Rather, it is proposed that fatigue in any form of exercise may form part of a regulated, anticipatory response co-ordinated in the subconscious brain. The ultimate goal of this regulation is to preserve homeostasis in all physiological systems during exercise, regardless of intensity or duration or the environmental conditions in which it is undertaken.

The MEL1 gene in Saccharomyces cerevisiae is required for the production of alpha-galactosidase and for the catabolism of melibiose. Production of alpha-galactosidase is induced by galactose or melibiose and repressed by glucose. Inducibility is controlled by the positive and negative regulatory proteins GAL4 and GAL80, respectively. We have cloned the MEL1 gene to study its transcriptional expression and regulation. Evidence is presented that the MEL1 gene encodes alpha-galactosidase and that mel0 is a naturally occurring allele which lacks the alpha-galactosidase-coding sequences. RNAs prepared from wild-type cells and from cells carrying either the noninducible gal4-2 or GAL80S-100 allele grown on three different carbon sources were examined by Northern hybridization analyses. In wild-type cells under noninducing conditions, such as growth on glycerol-lactic acid, the MEL1 transcript was detected at a basal level which was 1 to 2% of the fully induced level. The basal level of expression was diminished in cells carrying the gal4-2 mutant allele but not in cells carrying the GAL80S-100 allele. The basal and induced RNA levels are repressed by glucose. Size determinations of the MEL1 transcripts detected in glycerol-lactic acid- and galactose-grown cells provided no evidence for two distinct transcripts.

Probiotics are beneficial components of the microbiota that have been used for centuries because of the health benefits they confer to the host. Only recently, however, has the contribution of probiotics to modulation of immunological, respiratory, and gastrointestinal functions started to be fully appreciated and scientifically evaluated. Probiotics such as Escherichia coli Nissle 1917 and lactic acid bacteria are currently used to, or have been evaluated for use to, prevent or treat a range of intestinal maladies including inflammatory bowel disease, constipation, and colon cancer. Engineering these natural probiotics to produce immunomodulatory molecules may help to further increase the benefit to the host. In this article, we will discuss some of the mechanisms of action of probiotics as well as advances in the rational design of probiotics.

OBJECTIVE

Accumulation of asymmetrical dimethylarginine (ADMA) has been linked to endothelial dysfunction, and is an important risk factor for cardiovascular disease. Its elimination from the body is dependent on urinary excretion and degradation by the enzyme dimethylarginine dimethylaminohydrolase. This enzyme is highly expressed in the liver, and in rat studies a high net hepatic uptake of asymmetrical dimethylarginine was found. In critically ill patients, we investigated the relation between indicators of renal and hepatic dysfunction and plasma ADMA concentration, and tested the association between ADMA concentration and outcome.

METHODS

We prospectively collected blood samples from a cross-section of critically ill patients (n=52) with clinical evidence of dysfunction of more than two organs. We identified correlates of plasma ADMA concentration with laboratory values, organ failures score and outcome by univariate and multiple regression analyses.

RESULTS

In critically ill patients, plasma ADMA concentration was independently related to the presence of hepatic failure (b=0.334, 95% CI: 0.207-0.461; P<0.001), and to lactic acid (b=0.395, 95% CI: 0.230-0.560; P<0.001) and bilirubin (b=0.121, 95% CI: 0.031-0.212; P=0.009) concentration as markers of hepatic function. Twenty-one (40%) patients deceased during their ICU stay. In a logistic regression model, plasma ADMA ranked as the first and strongest predictor for outcome, with a 17-fold (95% CI: 3-100) increased risk for ICU death in patients who were in the highest quartile for ADMA.

CONCLUSIONS

In critically ill patients, plasma ADMA concentration is a strong and independent risk factor for ICU mortality, and hepatic dysfunction is the most prominent determinant of ADMA concentration in this population.

Clostridium difficile infection is increasing in both frequency and severity, with the emergence of new highly virulent strains highlighting the need for more rapid and effective methods of control. Here, we show that bacteriophage endolysin can be used to inhibit and kill C. difficile. The genome sequence of a novel bacteriophage that is active against C. difficile was determined, and the bacteriophage endolysin gene was subcloned and expressed in Escherichia coli. The partially purified endolysin was active against 30 diverse strains of C. difficile, and importantly, this group included strains of the major epidemic ribotype 027 (B1/NAP1). In contrast, a range of commensal species that inhabit the gastrointestinal tract, including several representatives of the clostridium-like Firmicutes, were insensitive to the endolysin. This endolysin provides a platform for the generation of both therapeutic and detection systems to combat the C. difficile problem. To investigate a method for the protected delivery and production of the lysin in the gastrointestinal tract, we demonstrated the expression of active CD27L endolysin in the lactic acid bacterium Lactococcus lactis MG1363.

Probiotic Lactobacillus can be used to reduce the colonization of pathogenic bacteria in food animals, and therefore reduce the risk of foodborne illness to consumers. As a model system, we examined the mechanism of protection conferred by Lactobacillus species to inhibit C. jejuni growth in vitro and reduce colonization in broiler chickens. Possible mechanisms for the reduction of pathogens by lactobacilli include: 1) stimulation of adaptive immunity; 2) alteration of the cecal microbiome; and, 3) production of inhibitory metabolites, such as organic acids. The Lactobacillus species produced lactic acid at concentrations sufficient to kill C. jejuni in vitro. We determined that lactic acid produced by Lactobacillus disrupted the membrane of C. jejuni, as judged by biophotonics. The spectral features obtained using Fourier-transform infrared (FT-IR) and Raman spectroscopy techniques were used to accurately predict bacterial viability and differentiate C. jejuni samples according to lactic acid treatment. FT-IR spectral features of C. jejuni and Lactobacillus grown in co-culture revealed that the metabolism was dominated by Lactobacillus prior to the killing of C. jejuni. Based on our results, the development of future competitive exclusion strategies should include the evaluation of organic acid production.