A series of zinc(II) and magnesium(II) alkoxides based upon a beta-diiminate ligand framework has been prepared. [(BDI-1)ZnO(i)Pr](2) [(BDI-1) = 2-((2,6-diisopropylphenyl)amido)-4-((2,6-diisopropylphenyl)imino)-2-pentene] exhibited the highest activity and stereoselectivity of the zinc complexes studied for the polymerization of rac- and meso-lactide to poly(lactic acid) (PLA). [(BDI-1)ZnO(i)()Pr](2) polymerized (S,S)-lactide to isotactic PLA without epimerization of the monomer, rac-lactide to heterotactic PLA (P(r) = 0.94 at 0 degrees C), and meso-lactide to syndiotactic PLA (P(r) = 0.76 at 0 degrees C). The polymerizations are living, as evidenced by the narrow polydispersities of the isolated polymers in addition to the linear nature of number average molecular weight versus conversion plots and monomer-to-catalyst ratios. The substituents on the beta-diiminate ligand exert a significant influence upon the course of the polymerizations, affecting both the degree of stereoselectivity and the rate of polymerization. Kinetic studies with [(BDI-1)ZnO(i)Pr](2) indicate that the polymerizations are first order with respect to monomer (rac-lactide) and 1.56 order in catalyst. Polymerization experiments with [(BDI-1)MgO(i)Pr](2) revealed that this complex is extremely fast for the polymerization of rac-lactide, polymerizing 500 equiv in 96% yield in less than 5 min at 20 degrees C.

A new kind of ultrasmall dissociable AuNR@PEG/PLGA vesicles (≈60 nm) (AuNR = gold nanorod; PEG = poly(ethylene glycol); PLGA = poly(lactic-co-glycolic acid)) assembled from small AuNRs (dimension: ≈8 nm × 2 nm) is reported. They exhibit several striking features: prolonged circulation and prominent tumor accumulation; rapid excretion from the body as AuNR@PEG after therapy; enhanced photoacoustic and photo thermal properties; and high photothermal cancer therapy efficacy.

The Warburg effect refers to the phenomenon whereby cancer cells avidly take up glucose and produce lactic acid under aerobic conditions. Although the molecular mechanisms underlying tumor reliance on glycolysis remains not completely clear, its inhibition opens feasible therapeutic windows for cancer treatment. Indeed, several small molecules have emerged by combinatorial studies exhibiting promising anticancer activity both in vitro and in vivo, as a single agent or in combination with other therapeutic modalities. Therefore, besides reviewing the alterations of glycolysis that occur with malignant transformation, this manuscript aims at recapitulating the most effective pharmacological therapeutics of its targeting. In particular, we describe the principal mechanisms of action and the main targets of 3-bromopyruvate, an alkylating agent with impressive antitumor effects in several models of animal tumors. Moreover, we discuss the chemo-potentiating strategies that would make unparalleled the putative therapeutic efficacy of its use in clinical settings.

The use of live microorganisms as an antigen delivery system is an effective means to elicit local immune responses and thus represents a promising strategy for mucosal vaccination. In this respect, lactic acid bacteria represent an original and attractive approach, as they are safe organisms that are used as food starters and probiotics. To determine whether an immune response could be elicited by intranasal delivery of recombinant lactobacilli, a Lactobacillus plantarum strain of human origin (NCIMB8826) was selected as the expression host. Cytoplasmic production of the 47-kDa fragment C of tetanus toxin (TTFC) was achieved at different levels depending on the plasmid construct. All recombinant strains proved to be immunogenic by the intranasal route in mice and able to elicit very high systemic immunoglobulin G (IgG1, IgG2b, and IgG2a) responses which correlated to the antigen dose. No significant differences in enzyme-linked immunosorbent assay IgG titers were observed when mice were immunized with live or mitomycin C-treated recombinant lactobacilli. Nevertheless, protection against the lethal effect of tetanus toxin was obtained only with the strains producing the highest dose of antigen and was greater following immunization with live bacteria. Significant TTFC-specific mucosal IgA responses were measured in bronchoalveolar lavage fluids, and antigen-specific T-cell responses were detected in cervical lymph nodes, both responses being higher in mice receiving a double dose of bacteria (at a 24-h interval) at each administration. These results demonstrate that recombinant lactobacilli can induce specific humoral (protective) and mucosal antibodies and cellular immune response against protective antigens upon nasal administration.

Dendritic cells are the most potent antigen-presenting cells (APC) and the most effective stimulators of primary T cell responses. Based on the strong influence of the APC on the immune response, we investigated cellular uptake of a biodegradable antigen delivery system, poly(D,L-lactic-co-glycolic acid) (PLGA) microspheres, at two sites of injection: intraperitoneal and intradermal. We hypothesized that a fluorescent probe, tetramethylrhodamine labeled dextran, loaded in PLGA microspheres would be taken up by APCs and thereby provide a means for studying cellular uptake of PLGA microspheres in vivo. Phagocytic load and cell phenotype were determined using flow cytometry and confocal laser scanning microscopy. The results revealed cellular uptake of tetramethylrhodamine dextran loaded PLGA microspheres at both injection sites. After intraperitoneal immunization, the predominant cell phagocytosing PLGA microspheres in the peritoneal cavity was the macrophage whereas the intradermal immunization resulted in uptake of PLGA microspheres by dendritic cells. Hence, these results suggest that the profile for cellular uptake varies with the site of injection. More importantly, this study provides direct and conclusive evidence of uptake of PLGA microspheres by the most potent APC, the dendritic cell.

Lactic acid bacteria (LAB) have proved to be effective mucosal delivery vehicles that overcome the problem of delivering functional proteins to the mucosal tissues. By the intranasal route, both live and killed LAB vaccine strains have been shown to elicit mucosal and systemic immune responses that afford protection against infectious challenges. To be effective via oral administration, frequent dosing over several weeks is required but new targeting and adjuvant strategies have clearly demonstrated the potential to increase the immunogenicity and protective immunity of LAB vaccines. Oral administration of Lactococcus lactis has been shown to induce antigen-specific oral tolerance (OT) to secreted recombinant antigens. LAB delivery is more efficient at inducing OT than the purified antigen, thus avoiding the need for purification of large quantities of antigen. This approach holds promise for new therapeutic interventions in allergies and antigen-induced autoimmune diseases. Several clinical and research reports demonstrate considerable progress in the application of genetically modified L. lactis for the treatment of inflammatory bowel disease (IBD). New medical targets are on the horizon, and the approval by several health authorities and biosafety committees of a containment system for a genetically modified L. lactis that secretes Il-10 should pave the way for new LAB delivery applications in the future.

The release kinetics of recombinant human bone morphogenetic protein-2 (rhBMP-2) loaded poly(dl-lactic-co-glycolic acid)/calcium phosphate cement (PLGA/Ca-P cement) composites were studied in vivo. RhBMP-2 was radiolabeled with (131)I and entrapped within PLGA microparticles or adsorbed onto the microparticle surface. PLGA microparticles were prepared of high molecular weight (HMW) PLGA (weight average molecular weight [M(w)] 49,100+/-1700) or low molecular weight (LMW) PLGA (M(w) 5,900+/-300) and used for preparation of 30:70 wt.% PLGA/Ca-P cement composite discs. Release of 131I-rhBMP-2 loaded composites was assessed by scintigraphic imaging according to a 2(2) two-level full factorial design in the rat ectopic model during four weeks. In vivo release kinetics varied among formulations. All formulations showed slow release without initial burst, and displayed a linear release from 3 to 28 days. Release of LMW entrapped rhBMP-2 composites (1.7+/-0.3%/day) was significantly faster than release from other formulations (p<0.01). After 28 days, retention within the composites was 65+/-5%, 75+/-4%, 50+/-4% and 70+/-6% of the initial rhBMP-2 for HMW entrapped, HMW adsorbed, LMW entrapped and LMW adsorbed rhBMP-2 composites, respectively. Release from the composite was probably slowed down by an interaction of rhBMP-2 and Ca-P cement after rhBMP-2 release from PLGA microparticles. We conclude that PLGA/Ca-P cement composites can be considered as sustained slow release vehicles and that the release and retention of rhBMP-2 can be modified according to the desired profile to a limited extent.

Most bacteria synthesize muramyl-pentapeptide peptidoglycan precursors ending with a D-alanyl residue (e.g., UDP-N-acetylmuramyl-L-Ala-gamma-D-Glu-L-Lys-D-Ala-D-Ala). However, it was recently demonstrated that other types of precursors, notably D-lactate-ending molecules, could be synthesized by several lactic acid bacteria. This particular feature leads to vancomycin resistance. Vancomycin is a glycopeptide antibiotic that blocks cell wall synthesis by the formation of a complex with the extremity of peptidoglycan precursors. Substitution of the terminal D-alanine by D-lactate reduces the affinity of the antibiotic for its target. Lactobacillus plantarum is a lactic acid bacterium naturally resistant to vancomycin. It converts most of the glycolytic pyruvate to L- and D-lactate by using stereospecific enzymes designated L- and D-lactate dehydrogenases, respectively. In the present study, we show that L. plantarum actually synthesizes D-lactate-ending peptidoglycan precursors. We also report the construction of a strain which is deficient for both D- and L-lactate dehydrogenase activities and which produces only trace amounts of D- and L-lactate. As a consequence, the peptidoglycan synthesis pathway is drastically affected. The wild-type precursor is still present, but a new type of D-alanine-ending precursor is also synthesized in large quantities, which results in a highly enhanced sensitivity to vancomycin.

It is recognised that ageing induces various changes to the human colonic microbiota. Most relevant is a reduction in bifidobacteria, which is a health-positive genus. Prebiotics, such as galacto-oligosaccharides (GOS), are dietary ingredients that selectively fortify beneficial gut microbial groups. Therefore, they have the potential to reverse the age-related decline in bifidobacteria and modulate associated health parameters. We assessed the effect of GOS mixture (Bimuno (B-GOS)) on gut microbiota, markers of immune function and metabolites in forty elderly (age 65-80 years) volunteers in a randomised, double-blind, placebo (maltodextrin)-controlled, cross-over study. The intervention periods consisted of 10 weeks with daily doses of 5·5 g/d with a 4-week washout period in between. Blood and faecal samples were collected for the analyses of faecal bacterial populations and immune and metabolic biomarkers. B-GOS consumption led to significant increases in bacteroides and bifidobacteria, the latter correlating with increased lactic acid in faecal waters. Higher IL-10, IL-8, natural killer cell activity and C-reactive protein and lower IL-1β were also observed. Administration of B-GOS to elderly volunteers may be useful in positively affecting the microbiota and some markers of immune function associated with ageing.

Targeted delivery of therapeutics to tumor neovasculature is potentially a powerful approach for selective cancer treatment. Integrins are heterodimeric transmembrane proteins involved in cell adhesion and cell signaling, and their expression is commonly upregulated in cancers and inflammatory diseases. The α(v)β(3) integrin is differentially upregulated on angiogenic endothelial cells as well as on many cancer cells. Here we demonstrate the differential targeting of cisplatin prodrug-encapsulated poly(d,l-lactic-co-glycolic acid)-block-polyethylene glycol (PLGA-PEG) nanoparticles (NPs) to the α(v)β(3) integrin on cancer cells using the cyclic pentapeptide c(RGDfK). Cisplatin is one of the most widely used anticancer drugs, and approaches that can improve its therapeutic index are of broad importance. The RGD-targeted Pt(IV)-encapsulated NPs displayed enhanced cytotoxicity as compared to cisplatin administered in its conventional dosage form in model prostate and breast cancer epithelial cells in vitro. Cytotoxicities were also elevated in comparison to those of previously reported systems, a small molecule Pt(IV)-RGD conjugate and a Pt(IV) nanoscale coordination polymer carrying RGD moieties. This result encouraged us also to evaluate the anticancer effect of the new construct in an animal model. The RGD-targeted PLGA-PEG NPs were more efficacious and better tolerated by comparison to cisplatin in an orthotopic human breast cancer xenograft model in vivo.

Lactic acid bacteria are food-grade microorganisms that are potentially good candidates for production of heterologous proteins of therapeutical or technological interest. We developed a model for heterologous protein secretion in Lactococcus lactis using the staphylococcal nuclease (Nuc). The effects on protein secretion of alterations in either (i) signal peptide or (ii) propeptide sequences were examined. (i) Replacement of the native Nuc signal peptide (SP(Nuc)) by that of L. lactis protein Usp45 (SP(Usp)) resulted in greatly improved secretion efficiency (SE). Pulse-chase experiments showed that Nuc secretion kinetics was better when directed by SP(Usp) than when directed by SP(Nuc). This SP(Usp) effect on Nuc secretion is not due to a better antifolding activity, since SP(Usp):Nuc precursor proteins display enzymatic activity in vitro, while SP(Nuc):Nuc precursor proteins do not. (ii) Deletion of the native Nuc propeptide dramatically reduces Nuc SE, regardless of which SP is used. We previously reported that a synthetic propeptide, LEISSTCDA, could efficiently replace the native Nuc propeptide to promote heterologous protein secretion in L. lactis (Y. Le Loir, A. Gruss, S. D. Ehrlich, and P. Langella, J. Bacteriol. 180:1895-1903, 1998). To determine whether the LEISSTCDA effect is due to its acidic residues, specific substitutions were introduced, resulting in neutral or basic propeptides. Effects of these two new propeptides and of a different acidic synthetic propeptide were tested. Acidic and neutral propeptides were equally effective in enhancing Nuc SE and also increased Nuc yields. In contrast, the basic propeptide strongly reduced both SE and the quantity of secreted Nuc. We have shown that the combination of the native SP(Usp) and a neutral or acidic synthetic propeptide leads to a significant improvement in SE and in the quantity of synthesized Nuc. These observations will be valuable in the production of heterologous proteins in L. lactis.

While lactic acid bacteria and bifidobacteria have been scientifically important for over a century, many of these are marketed today as probiotics and have become a valuable and rapidly expanding sector of the food market that is leading functional foods in many countries. The human gastro-intestinal tract with its various compartments and complex microbiota is the primary target of most of these functional foods containing lactic acid bacteria and bifidobacteria (LAB&B). In addition, their use as vectors for delivery of molecules with therapeutic value to the host via the intestinal tract is being studied. This review focuses on molecular approaches for the investigation of the diversity of lactic acid bacteria and bifidobacteria in the human intestine, as well as tracking of probiotic bacteria within this complex ecosystem. Moreover, methodologies to determine the viability of the lactic acid bacteria and bifidobacteria and molecular approaches to study the mechanisms by which they adapt, establish and interact with the human host via the digestive tract, are described.

The use of live bacteria to induce an immune response to itself or to a carried vaccine component is an attractive vaccine strategy. Advantages of live bacterial vaccines include their mimicry of a natural infection, intrinsic adjuvant properties and their possibility to be administered orally. Derivatives of pathogenic and non-pathogenic food related bacteria are currently being evaluated as live vaccines. However, pathogenic bacteria demands for attenuation to weaken its virulence. The use of bacteria as vaccine delivery vehicles implies construction of recombinant strains that contain the gene cassette encoding the antigen. With the increased knowledge of mucosal immunity and the availability of genetic tools for heterologous gene expression the concept of live vaccine vehicles gains renewed interest. However, administration of live bacterial vaccines poses some risks. In addition, vaccination using recombinant bacteria results in the release of live recombinant organisms into nature. This places these vaccines in the debate on application of genetically modified organisms. In this review we give an overview of live bacterial vaccines on the market and describe the development of new live vaccines with a focus on attenuated bacteria and food-related lactic acid bacteria. Furthermore, we outline the safety concerns and identify the hazards associated with live bacterial vaccines and try to give some suggestions of what to consider during their development.

L-cell-grown Chlamydia psittaci elementary bodies (EB) were rapidly phagocytized by mouse peritoneal macrophages in vitro. However, the intracellular fate of chlamydiae in macrophages appeared to be dependent on the multiplicity of infection (MOI), i.e., the EB-to-macrophage ratio, and the treatment of the EB. At an MOI of 1:1 or less, survival is maximal, and growth and multiplication of live, untreated chlamydiae did occur. In contrast, at a high MOI (100:1), survival of chlamydiae is reduced, as confirmed by release of 3H-labeled nucleic acid into the supernatant. At the high MOI, macrophage damage occurred that resulted in significant release of the lactic dehydrogenase, beginning 2 h postinfection. This immediated macrophage cytotoxicity as abolished by pretreatment of EB with heat (5 min at 56 degrees C) and was reduced about 50% by coating EB with homologous antibody. Pretreatment of the chlamydia with heat or opsonizing antibody provides increased uptake of EB by macrophages but may contribute to increased destruction of these obligate intracellular pathogens in professional phagocytic cells.

The majority of patients with MELAS (mitochondrial encephalomyophathy, lactic acidosis, stroke-like episodes) carry a heteroplasmic A3243G mutation in the mitochondrial tRNA(Leu(UUR)). The mutation prevents modification of the wobble U base, impairing translation at UUA and UUG codons; however, whether this results in amino acid misincorporation in the mitochondrial translation products remains controversial. We tested this hypothesis in homoplasmic mutant myoblasts isolated from a MELAS patient and investigated whether overexpression of the mitochondrial translation elongation factors could suppress the translation defect. Blue-Native gel electrophoretic analysis demonstrated an almost complete lack of assembly of respiratory chain complexes I, IV and V in MELAS myoblasts. This phenotype could be partially suppressed by overexpression of EFTu or EFG2 but not EFTs or EFG1. Despite the severity of the assembly defect, overall mitochondrial protein synthesis was only moderately affected, but some anomalously migrating translation products were present. Pulse-chase labeling showed reduced stability of all mitochondrial translation products consistent with the assembly defect. Labeling patterns of the translation products were similar with [(3)H]-leucine or [(3)H]-phenylalanine, showing that loss of the wobble U modification did not permit decoding of UUY codons; however, endoproteinase fingerprint analysis showed clear evidence of amino acid misincorporation in three polypeptides: CO III, CO II and ATP6. Taken together, these data demonstrate that the A3243G mutation produces both loss- and gain-of-function phenotypes, explaining the apparent discrepancy between the severity of the translation and respiratory chain assembly defects, and suggest a function for EFG2 in quality control of translation elongation.

Human umbilical cord mesenchymal stromal cells (hUCMSCs) have recently shown the capacity to differentiate into multiple cell lineages in all three embryonic germ layers. The osteogenic differentiation of hUCMSCs in monolayer culture has been reported, while the differentiation in three-dimensional biomaterials has not yet been reported for tissue-engineering applications. Thus, the aim of this study was to evaluate the feasibility of using hUCMSCs for bone tissue engineering. hUCMSCs were cultured in poly(L-lactic acid) (PLLA) scaffolds in osteogenic medium (OM) for 3 weeks, after which the scaffolds were exposed to several different media, including the OM, a mineralization medium (MM) and the MM with either 10 or 100 ng/ml insulin-like growth factor (IGF)-1. The osteogenic differentiation was confirmed by the up-regulation of Runx2 and OCN, calcium quantification and bone histology. Switching from the OM to the MM promoted collagen synthesis and calcium content per cell, while continuing in the OM retained more cells in the constructs and promoted higher osteogenic gene expression. The addition of IGF-1 into the MM had no effect on cell proliferation, differentiation and matrix synthesis. In conclusion, hUCMSCs show significant potential for bone tissue engineering and culturing in the OM throughout the entire period is beneficial for osteogenic differentiation of these cells.

In vaccination programmes in which large numbers of subjects are involved, the oral route of administration is more convenient as compared to the more frequently used parenteral route. This is particularly relevant when vaccines are to be applied in less industrialized countries. Lactic acid bacteria in general and strains of Lactobacillus in particular have a variety of properties which make them attractive candidates for oral vaccination purposes, e.g. GRAS status, adjuvant properties, mucosal adhesive properties and low intrinsic immunogenicity. An overview is given of current research aimed at unravelling the relationship between structure and properties of surface proteins of lactobacilli and in vivo colonization, in particular of species capable of adhering to epithelial cells in vitro. Secondly, the state of the art will be discussed with respect to antigen presentation by lactic acid bacteria. Finally, some preliminary immunological data of recombinant lactic acid bacterial strains expressing antigens from pathogens will be presented.

The influence of pH adjusted with lactic acid or HCl or sodium chloride concentration on survival or growth of Escherichia coli O157:H7 in Trypticase soy broth (TSB) was determined. Studies also determined the fate of E. coli O157:H7 during the production and storage of fermented, dry sausage. The organism grew in TSB containing less than or equal to 6.5% NaCl or at a pH of 4.5 to 9.0, adjusted with HCl. When TSB was acidified with lactic acid, the organism grew at pH 4.6 but not at pH 4.5. A commercial sausage batter inoculated with 4.8 x 10(4) E. coli O157:H7 per g was fermented to pH 4.8 and dried until the moisture/protein ratio was less than or equal to 1.9:1. The sausage chubs were then vacuum packaged and stored at 4 degrees C for 2 months. The organism survived but did not grow during fermentation, drying, or subsequent storage at 4 degrees C and decreased by about 2 log10 CFU/g by the end of storage. These studies reveal the importance of using beef containing low populations or no E. coli O157:H7 in sausage batter, because when initially present at 10(4) CFU/g, this organism can survive fermentation, drying, and storage of fermented sausage regardless of whether an added starter culture was used.

The complete gastrointestinal (GI) tract of humans is colonised soon after birth by a myriad of microbial species with a characteristic distribution depending on the location. GI-tract ecology has been experiencing a revival due to the development of molecular techniques, especially those based on 16S RNA (zRNA) genes. A richer ecosystem than previously imagined of novel species is being discovered that is significantly influenced by our host genotype. Special attention has been focused on the bifidobacteria and the lactic acid bacterial (LAB) populations, both those that are naturally present within this complex ecosystem and those that are ingested as probiotics in functional foods. Overall this interest stems from a increasing awareness of interplay between microflora, diet and the health of the host, and is further stimulated by an increasing incidence of gastrointestinal illnesses, and atopy. Substantial documentation of benefits to host health has especially distinguished the LAB for multidisciplinary research aimed to determine the molecular mechanisms involved. Recent advances in molecular technologies, including high-throughput genomics-based approaches, can significantly advance our understanding of the microbe--diet--host interactions and offer valuable information for design and application of health-targeted microbes.

We have previously reported the efficient osteogenic differentiation of human embryonic stem cells (hESCs) by co-culture with primary human bone-derived cells (hPBDs) without the use of exogenous factors. In the present study, we explored whether osteogenic cells derived from hESCs (OC-hESCs) using the previously reported method would be capable of regenerating bone tissue in vivo. A three-dimensional porous poly(d,l-lactic-co-glycolic acid)/hydroxyapatite composite scaffold was used as a cell delivery vehicle. In vivo implantation of OC-hESC-seeded scaffolds showed significant bone formation in the subcutaneous sites of immunodeficient mice at 4 and 8 weeks after implantation (n=5 for each time point). Meanwhile, implantation of the control no cell-seeded scaffolds or human dermal fibroblast-seeded scaffolds did not show any new bone formation. In addition, the presence of BMP-2 (1 microg/scaffold) enhanced new bone tissue formation in terms of mineralization and the expression of bone-specific genetic markers. According to FISH analysis, implanted OC-hESCs remained in the regeneration sites, which suggested that the implanted cells participated in the formation of new bone. In conclusion, OC-hESCs successfully regenerated bone tissue upon in vivo implantation, and this regeneration can be further enhanced by the administration of BMP-2. These results suggest the clinical feasibility of OC-hESCs as a good alternative source of cells for bone regeneration.

BSA was entrapped in particles of different sizes (200, 500 and 1000 nm) prepared from poly(D,L-lactic-co-glycolic) acid by a double emulsion method. The particles were given, either intranasally, orally or subcutaneously, to Balb/c mice and the serum IgG, IgG1 and IgG2a response elicited was compared to that obtained by the subcutaneous administration of either free antigen, free antigen emulsified 1:1 with Freund's Complete Adjuvant (FCA), or free antigen administered with Al(OH)(3). The administration of 1000 nm particles generally elicited a higher serum IgG response than that obtained with the administration of 500 or 200 nm sized nanospheres, the immune response for 500 nm particles being similar than that obtained with 200 nm by the subcutaneous and the oral route, and higher by the intranasal route. PLGA nanoparticles can elicit serum IgG2a responses by the three routes studied. No significant differences on the serum IgG2a/IgG1 ratios were found after the subcutaneous, the oral and the intranasal administration of the different spheres but those were in general higher compared to the administration of either free antigen or free antigen adsorbed to alum. The route of administration influences the serum IgG2a/IgG1 ratio after the administration of free antigen, but not after the administration of the particles. Therefore, differences on the total serum IgG response induced by particles of different sizes do not result in differences on the IgG1 or IgG2a-type immune responses, suggesting that the antigen processing and presentation is similar in all cases tested for PLGA particles.

Recent phosphoproteomics studies of several bacterial species have firmly established protein phosphorylation on Ser/Thr/Tyr residues as a PTM in bacteria. In particular, our recent reports on the Ser/Thr/Tyr phosphoproteomes of bacterial model organisms Bacillus subtilis and Escherichia coli detected over 100 phosphorylation events in each of the bacterial species. Here we extend our analyses to Lactococcus lactis, a lactic acid bacterium widely employed by the food industry, in which protein phosphorylation at Ser/Thr/Tyr residues was barely studied at all. Despite the lack of almost any prior evidence of Ser/Thr/Tyr protein phosphorylation in L. lactis, we identified a phosphoproteome of a size comparable to that of E. coli and B. subtilis, with 73 phosphorylation sites distributed over 63 different proteins. The presence of several multiply phosphorylated proteins, as well as over-representation of phosphothreonines seems to be the distinguishing features of the L. lactis phosphoproteome. Evolutionary comparison and the conservation of phosphorylation sites in different bacterial organisms indicate that a majority of the detected phosphorylation sites are species-specific, and therefore have probably co-evolved with the adaptation of the bacterial species to their present-day ecological niches.

Cultured astroglial cells are able to utilize the monosaccharides glucose, mannose, or fructose as well as the sugar alcohol sorbitol as energy fuel. Astroglial uptake of the aldoses is carrier-mediated, whereas a non-saturable transport mechanism is operating for fructose and sorbitol. The first metabolic step for all sugars, including fructose being generated by enzymatic oxidation of sorbitol, is phosphorylation by hexokinase. Besides glucose only mannose may serve as substrate for build-up of astroglial glycogen. Whereas glycogen synthase appears to be present in astrocytes as well as neurons, the exclusive localization of glycogen phosphorylase in astrocytes and ependymal cells of central nervous tissue correlates well with the occurrence of glycogen in these cells. The identification of lactic acid rather than glucose as degradation product of astroglial glycogen appears to render the presence of glucose-6-phosphatase in cultured astrocytes an enigma. The colocalization of pyruvate carboxylase, phosphenolpyruvate carboxykinase and fructose-1,6-bisphosphatase points to astrocytes as being the gluconeogenic cell type of the CNS.

Retinal progenitor cells (RPCs) are self-renewing cells capable of differentiating into the different retinal cell types including photoreceptors, and they have shown promise as a source of replacement cells in experimental models of retinal degeneration. We hypothesized that a biodegradable polymer scaffold could deliver these cells to the subretinal space in a more organized manner than bolus injections, while also providing the graft with laminar organization and structural guidance channels. We fabricated highly porous scaffolds from blends of poly(L-lactic acid) and poly(lactic-co-glycolic acid) using a variety of techniques to produce pores oriented normal to the plane of the scaffold. RPCs were seeded on the polymer scaffolds and cultured for 14 days. Seeded scaffolds were then either fixed for characterization or used in an explant or in vivo rat model. The scaffolds were fully covered by RPCs in 3 days. Attachment of RPCs to the polymer scaffold was associated with down-regulation of immature markers and up-regulation of markers of differentiation. This suggests that the scaffold may promote differentiation of RPCs. The seeded cells elaborated cellular processes and aligned in the scaffold in conjunction with degenerating retinal explants. The cells also exhibited morphologies consistent with photoreceptors including a high degree of polarization of the cells. This data suggests that the scaffold may be a means to assist in the promotion of photoreceptor phenotypes. Implantation of the seeded scaffold into the rat eye is associated with increased RPC survival. Taken together, these data suggest that these polymer scaffolds provide a useful means for delivering RPCs to the subretinal space and may assist in the formation of retinal cell phenotypes, although it is clear that more cues are needed to direct the differentiation of RPCs into functional photoreceptors.