We evaluated anti-Campylobacter jejuni activity among >1,200 isolates of different lactic acid bacteria. Lactobacillus salivarius strain NRRL B-30514 was selected for further study. The cell-free, ammonium sulfate precipitate from the broth culture was termed the crude antimicrobial preparation. Ten microliters of the crude preparation created a zone of C. jejuni growth inhibition, and growth within the zone resumed when the crude preparation was preincubated with proteolytic enzymes. Bacteriocin OR-7, derived from this crude preparation, was further purified using ion-exchange and hydrophobic-interaction chromatography. The determined amino acid sequence was consistent with class IIa bacteriocins. Interestingly, OR-7 had sequence similarity, even in the C-terminal region, to acidocin A, which was previously identified from L. acidophilus and had activity only to gram-positive bacteria, whereas OR-7 had activity to a gram-negative bacterium. Bacteriocin activity was stable following exposure to 90 degrees C for 15 min, also consistent with these types of antibacterial peptides. The purified protein was encapsulated in polyvinylpyrrolidone and added to chicken feed. Ten day-of-hatch chicks were placed in each of nine isolation units; two groups of birds were challenged with each of four C. jejuni isolates (one isolate per unit). At 7 days of age, one group of birds was treated with bacteriocin-emended feed for 3 days, and one group was left untreated. At 10 days of age, the birds were sacrificed and the challenge strain was enumerated from the bird cecal content. Bacteriocin treatment consistently reduced colonization at least one millionfold compared with levels found in the untreated groups. Nonchallenged birds were never colonized by C. jejuni. Bacteriocin from L. salivarius NRRL B-30514 appears potentially very useful to reduce C. jejuni in poultry prior to processing.

General aspects of biodegradable microspheres prepared from natural and synthesized polymers used in drug delivery systems are reviewed first from various viewpoints: characteristics of biodegradable polymers (physicochemical properties, bioerosion mechanism, biocompatibility), preparation method for the microspheres, drug release from parenteral products and briefly nonparenteral products. The relationship between release pattern and pharmacological activity of therapeutic peptides and proteins and rational controlled release design are also discussed. In the latter half, successful sustained release depot formulations of peptides, leuprorelin acetate, and thyrotropin-releasing hormone (TRH), utilizing poly(lactic acid) (PLA) and poly(lactic/glycolic acid) (PLGA) microspheres are reviewed with respect to preparation, drug release, biocompatibility, pharmacological effects, and results of clinical studies. Thereafter, studies on antitumor therapy by chemoembolization using PLGA microspheres containing an angiogenesis inhibitor (TNP-470) are described as an example of targeted drug delivery with biodegradable microspheres.

Exposure to oxygen deficits is more widespread in biological systems than is commonly believed. Until recently, the general perception of anaerobic metabolism was often limited to the induction of alcoholic or lactic acid fermentation as the sole biochemical response to hypoxia/anoxia. Developments in the physiology, biochemistry, and molecular biology of anaerobic responses in invertebrates, lower plants, and higher plants have demonstrated that, depending upon the species, anaerobic metabolism may encompass much more than simple glycolytic metabolism. Here, recent progress in elucidating the mechanism(s) determining tolerance versus intolerance to anaerobic environments in higher plants is discussed, drawing most heavily on experimental systems using seeds or seedlings.

A novel method was developed to prepare three-dimensional structures with desired shapes used as templates for cell transplantation. The produced biomaterials are highly porous with large surface/volume and provide the necessary space for attachment and proliferation of the transplanted cells. The processing technique calls for the formation of a composite material with nonbonded fibers embedded in a matrix followed by thermal treatment and the selective dissolution of the matrix. To evaluate the technique, poly(glycolic acid) (PGA) fiber meshes were bonded using poly(L-lactic acid) (PLLA) as a matrix. The bonded structures were highly porous with values of porosity up to 0.81 and area/volume ratios as high as 0.05 micron-1.

Some lactic acid bacteria (LAB) secrete a polysaccharide polymer. This extracellular polysaccharide, or "exopolysaccharide" (EPS), is economically important because it can impart functional effects to foods and confer beneficial health effects. LAB have a "Generally Recognized As Safe" (GRAS) classification and are likely candidates for the production of functional EPSs. Current challenges are to improve the productivity of EPSs from LAB and to produce EPSs of a structure and size that impart the desired functionality. The engineering of improvements in these properties will depend on a deep understanding of the EPS biosynthetic metabolism and of how the structure of EPSs relates to a functional effect when incorporated into a food matrix.

The purpose of this study was to evaluate the physiologic responses to endurance training in patients with moderate to severe airflow obstruction by specifically looking at changes in skeletal muscle enzymatic activities. Eleven patients (age = 65 +/- 7 yr, mean +/- SD, FEV1 = 36 +/- 11% of predicted value, range = 24 to 54%) were evaluated before and after an endurance training program. Each evaluation included a percutaneous biopsy of the vastus lateralis and a stepwise exercise test on an ergocycle up to his/her maximal capacity. VE, VO2, VcO2, and serial arterial lactic acid concentration were measured during the exercise test. The activity of two oxidative enzymes, citrate synthase (CS) and 3-hydroxyacyl-CoA dehydrogenase (HADH), and of three glycolytic enzymes, lactate dehydrogenase, hexokinase, and phosphofructokinase was determined. The training consisted of 30-min exercise sessions on a calibrated ergocycle, 3 times a week for 12 wk. The aerobic capacity was severely reduced at baseline (VO2max = 54 +/- 12% of predicted) and increased by 14% after training (p < 0.05). For an identical exercise workload, there was a significant reduction in VE (34.5 +/- 10.0 versus 31.9 +/- 9.0 L/min, p < 0.05) and in arterial lactic acid concentration (3.4 +/- 1.3 versus 2.8 +/- 0.9 mmol/L, p < 0.01) after training. The lactate threshold also increased after training (p < 0.01) while the activity of the three glycolytic enzymes was similar at the two evaluations. In contrast, the activity of CS and HADH increased significantly after training (22.3 +/- 3.5 versus 25.8 +/- 3.8 mumol/min/g muscle for CS, p < 0.05, and 5.5 +/- 2.9 versus 7.7 +/- 2.5 mumol/min/g for HADH, p < 0.01). A significant inverse relationship was found between the percent changes in the activity of CS and HADH, and the percent changes in arterial lactic acid during exercise (p = 0.01). We conclude that endurance training can reduce exercise-induced lactic acidosis and improve skeletal muscle oxidative capacity in patients with moderate to severe chronic obstructive pulmonary disease (COPD).

BACKGROUND

Hyperoxaluria is a major risk factor for renal stones, and in most cases, it appears to be sustained by increased dietary load or increased intestinal absorption. Previous studies have shown that components of the endogenous digestive microflora, in particular Oxalobacter formigenes, utilize oxalate in the gut, thus limiting its absorption. We tested the hypothesis of whether oxaluria can be reduced by means of reducing intestinal absorption through feeding a mixture of freeze-dried lactic acid bacteria.

METHODS

Six patients with idiopathic calcium-oxalate urolithiasis and mild hyperoxaluria (>40 mg/24 h) received daily a mixture containing 8 x 10(11) freeze-dried lactic acid bacteria (L. acidophilus, L. plantarum, L. brevis, S. thermophilus, B. infantis) for four weeks. The 24-hour urinary excretion of oxalate was determined at the end of the study period and then one month after ending the treatment. The ability of bacteria to degrade oxalate and grow in oxalate-containing media, and the gene expression of Ox1T, an enzyme that catalyzes the transmembrane exchange of oxalate, also were investigated.

RESULTS

The treatment resulted in a great reduction of the 24-hour excretion of oxalate in all six patients enrolled. Mean levels +/- SD were 33.5 +/- 15.9 mg/24 h at the end of the study period and 28.3 +/- 14.6 mg/24 h one month after treatment was interrupted compared with baseline values of 55.5 +/- 19.6 mg/24 h (P < 0.05). The treatment was associated with a strong reduction of the fecal excretion of oxalate in the two patients tested. Two bacterial strains among those used for the treatment (L. acidophilus and S. thermophilus) proved in vitro to degrade oxalate effectively, but their growth was somewhat inhibited by oxalate. One strain (B. infantis) showed a quite good degrading activity and grew rapidly in the oxalate-containing medium. L. plantarum and L. brevis showed a modest ability to degrade oxalate even though they grew significantly in oxalate-containing medium. No strain expressed the Ox1T gene.

CONCLUSIONS

The urinary excretion of oxalate, a major risk factor for renal stone formation and growth in patients with idiopathic calcium-oxalate urolithiasis, can be greatly reduced with treatment using a high concentration of freeze-dried lactic acid bacteria. We postulate that the biological manipulation of the endogenous digestive microflora can be a novel approach for the prevention of urinary stone formation.

BACKGROUND

Among the potent anticancer agents, curcumin has been found to be very efficacious against many different types of cancer cells. However, the major disadvantage associated with the use of curcumin is its low systemic bioavailability when administered orally due to its poor aqueous solubility. Our present work investigated the efficiency of encapsulation of curcumin in poly (lactic-coglycolic acid) (PLGA) nanospheres using solid/oil/water emulsion solvent evaporation method.

METHODS

The nanospheres were formulated and then characterized for percent yield, encapsulation efficiency, surface morphology, particle size, drug distribution studies, drug polymer interaction studies and in vitro drug release profiles.

RESULTS

Our studies showed the successful formation of smooth and spherical curcumin-loaded PLGA nanospheres, with an encapsulation efficiency of 90.88+/-0.14%. The particle size distribution showed a range of 35 nm to 100 nm, with the mean particle size being 45 nm. Evaluation of these curcumin-loaded nanospheres was carried out in prostate cancer cell lines. Results showed robust intracellular uptake of the nanospheres in the cells. Cell viability studies revealed that the curcumin-loaded nanospheres were able to exert a more pronounced effect on the cancer cells as compared to free curcumin.

CONCLUSIONS

Our studies achieved successful formulation of curcumin loaded PLGA nanospheres, thus indicating that nanoparticle-based formulation of curcumin has high potential as an adjuvant therapy for clinical application in prostate cancer.

The current taxonomy of probiotic lactic acid bacteria is reviewed with special focus on the genera Lactobacillus, Bifidobacterium and Enterococcus. The physiology and taxonomic position of species and strains of these genera were investigated by phenotypic and genomic methods. In total, 176 strains, including the type strains, have been included. Phenotypic methods applied were based on biochemical, enzymatical and physiological characteristics, including growth temperatures, cell wall analysis and analysis of the total soluble cytoplasmatic proteins. Genomic methods used were pulsed field gel electrophoresis (PFGE), randomly amplified polymorphic DNA-PCR (RAPD-PCR) and DNA-DNA hybridization for bifidobacteria. In the genus Lactobacillus the following species of importance as probiotics were investigated: L. acidophilus group, L. casei group and L. reuteri/L. fermentum group. Most strains referred to as L. acidophilus in probiotic products could be identified either as L. gasseri or as L. johnsonii, both members of the L. acidophilus group. A similar situation could be shown in the L. casei group, where most of the strains named L. casei belonged to L. paracasei subspp. A recent proposal to reject the species L. paracasei and to include this species in the restored species L. casei with a neotype strain was supported by protein analysis. Bifidobacterium spp. strains have been reported to be used for production of fermented dairy and recently of probiotic products. According to phenotypic features and confirmed by DNA-DNA hybridization most of the bifidobacteria strains from dairy origin belonged to B. animalis, although they were often declared as B. longum by the manufacturer. From the genus Enterococcus, probiotic Ec. faecium strains were investigated with regard to the vanA-mediated resistance against glycopeptides. These unwanted resistances could be ruled out by analysis of the 39 kDa resistance protein. In conclusion, the taxonomy and physiology of probiotic lactic acid bacteria can only be understood by using polyphasic taxonomy combining morphological, biochemical and physiological characteristics with molecular-based phenotypic and genomic techniques.

The present study aims to evaluate the probiotic potential of lactic acid bacteria (LAB) isolated from naturally fermented olives and select candidates to be used as probiotic starters for the improvement of the traditional fermentation process and the production of newly added value functional foods. Seventy one (71) lactic acid bacterial strains (17 Leuconostoc mesenteroides, 1 Ln. pseudomesenteroides, 13 Lactobacillus plantarum, 37 Lb. pentosus, 1 Lb. paraplantarum, and 2 Lb. paracasei subsp. paracasei) isolated from table olives were screened for their probiotic potential. Lb. rhamnosus GG and Lb. casei Shirota were used as reference strains. The in vitro tests included survival in simulated gastrointestinal tract conditions, antimicrobial activity (against Listeria monocytogenes, Salmonella Enteritidis, Escherichia coli O157:H7), Caco-2 surface adhesion, resistance to 9 antibiotics and haemolytic activity. Three (3) Lb. pentosus, 4 Lb. plantarum and 2 Lb. paracasei subsp. paracasei strains demonstrated the highest final population (>8 log cfu/ml) after 3 h of exposure at low pH. The majority of the tested strains were resistant to bile salts even after 4 h of exposure, while 5 Lb. plantarum and 7 Lb. pentosus strains exhibited partial bile salt hydrolase activity. None of the strains inhibited the growth of the pathogens tested. Variable efficiency to adhere to Caco-2 cells was observed. This was the same regarding strains' susceptibility towards different antibiotics. None of the strains exhibited β-haemolytic activity. As a whole, 4 strains of Lb. pentosus, 3 strains of Lb. plantarum and 2 strains of Lb. paracasei subsp. paracasei were found to possess desirable in vitro probiotic properties similar to or even better than the reference probiotic strains Lb. casei Shirota and Lb. rhamnosus GG. These strains are good candidates for further investigation both with in vivo studies to elucidate their potential health benefits and in olive fermentation processes to assess their technological performance as novel probiotic starters.

The use of Gram-positive bacteria for heterologous protein production proves to be a useful choice due to easy protein secretion and purification. The lactic acid bacterium Lactococcus lactis emerges as an attractive alternative to the Gram-positive model Bacillus subtilis. Here, we review recent work on the expression and secretion systems available for heterologous protein secretion in L. lactis, including promoters, signal peptides and mutant host strains known to overcome some bottlenecks of the process. Among the tools developed in our laboratory, inactivation of HtrA, the unique housekeeping protease at the cell surface, or complementation of the Sec machinery with B. subtilis SecDF accessory protein each result in the increase in heterologous protein yield. Furthermore, our lactococcal expression/secretion system, using both P(Zn)zitR, an expression cassette tightly controlled by environmental zinc, and a consensus signal peptide, SP(Exp4), allows efficient production and secretion of the staphylococcal nuclease, as evidenced by protein yields (protein amount/biomass) comparable to those obtained using NICE or P170 expression systems under similar laboratory conditions. Finally, the toolbox we are developing should contribute to enlarge the use of L. lactis as a protein cell factory.

Nanoparticles formulated from biodegradable polymers such as poly(lactic acid) (PLA) and poly(lactide-co-glycolide) (PLGA) are being extensively investigated as non-viral gene delivery systems due to their controlled release characteristics and biocompatibility. PLGA nanoparticles for DNA delivery are mainly formulated by an emulsion-solvent evaporation technique using PVA as a stabilizer generating negatively charged particles and heterogeneous size distribution. The objective of the present study was to formulate cationically modified PLGA nanoparticles with defined size and shape that can efficiently bind DNA. An Emulsion-diffusion-evaporation technique to make cationic nanospheres composed of biodegradable and biocompatible co-polyester PLGA has been developed. PVA-chitosan blend was used to stabilize the PLGA nanospheres. The nanospheres were characterized by atomic force microscopy (AFM), photon-correlation spectroscopy (PCS), and Fourier transform infrared spectroscopy (FTIR). Zeta potential and gel electrophoresis studies were also performed to understand the surface properties of nanospheres and their ability to condense negatively charged DNA. The designed nanospheres have a zeta potential of 10mV at pH 7.4 and size under 200nm. From the gel electrophoresis studies we found that the charge on the nanospheres is sufficient to efficiently bind the negatively charged DNA electrostatically. These cationic PLGA nanospheres could serve as potential alternatives of the existing negatively charged nanoparticles.

Impaired metabolism of short-chain fatty acids, as well as a modified fecal ionogram, have been reported in ulcerative colitis. Fecal water samples from 62 patients with ulcerative colitis were analyzed in the present investigation to evaluate changes in SCFAs and lactic acid in relation to activity and severity of disease. Short-chain fatty acid levels were high in quiescent and mild disease (162.6 +/- 63.6 and 147.8 +/- 63.2 mM/L, respectively), but significantly decreased in the severe form (64.7 +/- 46.9 mM/L). Lactate showed a progressive increase from mild colitis (3.0 +/- 1.8 mM/L) to severe colitis (21.4 +/- 18.6 mM/L). It thus appears that mild colitis displayed a fecal pattern characterized by normal pH and bicarbonate, slightly impaired electrolyte handling, high short-chain fatty acid values, and only moderately increased lactate. Severe colitis, on the other hand, was characterized by low fecal pH, bicarbonate, and potassium, high sodium and chloride, low short-chain fatty acid levels, and very high lactate levels. A critical lowering of intraluminal pH, which shifts bacterial metabolism from short-chain fatty acid to lactate production, may be responsible for the intraluminal pooling of lactate.

This study was carried out to investigate the relationship between the conduction velocity of the vagal afferents arising from the rat lungs and their sensitivities to capsaicin, other chemical irritants, and lung inflation. We recorded single-unit activities of vagal pulmonary afferents (n = 205) in anesthetized, open-chest rats, and distinguished C fibers (conduction velocity < 2 m/sec) from myelinated afferents; the latter group was further classified into rapidly adapting pulmonary receptors (RARs) and slowly adapting pulmonary stretch receptors (SARs) on the basis of their adaptation indexes to lung inflation. Right-atrial injection of capsaicin (1 microg/kg) evoked an abrupt and intense stimulatory effect in 88.9% (64/72) of the pulmonary C fibers tested, but only a mild stimulation in 6.3% (3/48) of the RARs and none of the SARs. Other inhaled and injected chemical stimulants (e.g., cigarette smoke, lactic acid) activated 68.9% (42/61) of the pulmonary C fibers. The same chemical irritants exerted a mild stimulatory effect in only 14.5% (8/55) of the RARs; this subgroup of RARs exhibited a low or no baseline activity, and half of them were located near the hilum. Chemical stimulants had little or no effect on SARs. The response of pulmonary C fibers to lung inflation (tracheal pressure = 30 cm H2O) was not only extremely weak, but also showed a longer onset latency and an irregular pattern. In a sharp contrast, lung inflation evoked rapid and vigorous discharges in both RARs and SARs. In conclusion, C fibers are the primary type of chemosensitive vagal pulmonary afferents in rat lungs.

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, environmental 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.

Human sweat samples were chemically fractionated into acid and non-acid components. The most abundant volatile compounds present in the fractions were identified by linked gas chromatography mass spectrometry. The acid fractions were found to be composed of a range of twenty aliphatic and three aromatic carboxylic acids ranging, on average, from 0.02 to 20 micrograms per ml of sweat sampled. Non-acid fractions were found to contain: 6-methyl-5-hepten-2-one, 1-octen-3-ol, decanal, benzyl alcohol, dimethylsulphone, phenylethanol, phenol and 4-methylphenol, collectively amounting to 0.1 and 3 micrograms per ml of sweat. The major component of sweat was found to be L-lactic acid which constituted from 1 to 5 mg/ml. Using the intact antennae of the anthropophilic malaria vector mosquito Anopheles gambiae Giles, the peripheral olfactory activities of compounds identified in the sweat fractions were investigated by electroantennography (EAG). Short-chain saturated carboxylic acids, methanoic, ethanoic, propanoic, butanoic, pentanoic and hexanoic acids were found to elicit significantly larger EAG responses than longer chain saturated carboxylic acids from female An.gambiae. For a given dose the largest amplitude EAG response was elicited by methanoic acid. Pentanoic acid elicited larger EAG responses than either butanoic or hexanoic acids. Two non-acidic compounds, 1-octen-3-ol and 4-methylphenol, were found to elicit significant dose-dependent EAG responses from female An.gambiae. 1-Octen-3-ol elicited larger EAG responses than 4-methylphenol for a given dose, but both compounds elicited smaller EAG responses than the same dose of C1-C6 straight-chain aliphatic carboxylic acids. The possible behavioural significance of the EAG-active compounds identified in human sweat samples is discussed.

We describe a general method for incorporating target ligands into the surface of biocompatible polyester poly(lactic-co-glycolic acid) (PLGA) 50/50 materials using fatty acids. Avidin-fatty acid conjugates were prepared and efficiently incorporated into PLGA. Avidin was chosen as an adaptor protein to facilitate the attachment of a variety of biotinylated ligands. We show that fatty acid preferentially associates with the hydrophobic PLGA matrix, rather than the external aqueous environment, facilitating a prolonged presentation of avidin over several weeks. We successfully applied this approach in both microspheres encapsulating a model protein, bovine serum albumin, and PLGA scaffolds fabricated by a salt-leaching method. Because of its ease, generality and flexibility, this strategy promises widespread utility in modifying the surface of PLGA-based materials for applications in drug delivery and tissue engineering.

Lactic acid bacteria (LAB) are frequently encountered inhabitants of the human intestinal tract. A protective layer of mucus covers the epithelial cells of the intestine, offering an attachment site for these bacteria. In this study bioinformatics tools were used to identify and characterize proteins containing one type of mucus-binding domain, called MUB, that is postulated to play an important role in the adherence of LAB to this mucus layer. By searching in all protein databases 48 proteins containing at least one of these MUB domains in nine LAB species were identified. These MUB domains varied in size, ranging from approximately 100 to more than 200 residues per domain. Complete MUB domains were found exclusively in LAB. The number of MUB domains present in a single protein varied from 1 to 15. In some cases, orthologous proteins in closely related species contained a different number of domains, indicating that repeats of the domain undergo rapid duplication and deletion. Proteins containing the MUB domain were often encoded by gene clusters that encode multiple extracellular proteins. In addition to one or more copies of the MUB domain, many of these proteins contained other domains that are predicted to be involved in binding to and degradation of extracellular components. These findings strongly suggest that the MUB domain is an LAB-specific functional unit that performs its task in various domain contexts and could fulfil an important role in host-microbe interactions in the gastrointestinal tract.

Streptococci and veillonellae occur in mixed-species colonies during formation of early dental plaque. One factor hypothesized to be important in assembly of these initial communities is coaggregation (cell-cell recognition by genetically distinct bacteria). Intrageneric coaggregation of streptococci occurs when a lectin-like adhesin on one streptococcal species recognizes a receptor polysaccharide (RPS) on the partner species. Veillonellae also coaggregate with streptococci. These genera interact metabolically; lactic acid produced by streptococci is a carbon source for veillonellae. To transpose these interactions from undisturbed dental plaque to an experimentally tractable in vitro biofilm model, a community consisting of RPS-bearing streptococci juxtaposed with veillonellae was targeted by quantum dot-based immunofluorescence and then micromanipulated off the enamel surface and cultured. Besides the expected antibody-reactive cell types, a non-antibody-reactive streptococcus invisible during micromanipulation was obtained. The streptococci were identified as Streptococcus oralis (RPS bearing) and Streptococcus gordonii (adhesin bearing). The veillonellae could not be cultivated; however, a veillonella 16S rRNA gene sequence was amplified from the original isolation mixture, and this sequence was identical to the sequence of the previously studied organism Veillonella sp. strain PK1910, an oral isolate in our culture collection. S. oralis coaggregated with S. gordonii by an RPS-dependent mechanism, and both streptococci coaggregated with PK1910, which was used as a surrogate during in vitro community reconstruction. The streptococci and strain PK1910 formed interdigitated three-species clusters when grown as a biofilm using saliva as the nutritional source. PK1910 grew only when streptococci were present. This study confirms that RPS-mediated intrageneric coaggregation occurs in the earliest stages of plaque formation by bringing bacteria together to create a functional community.

Rice (Oryza sativa) accumulates prolamins and glutelins as storage proteins. The latter storage protein is synthesized on the endoplasmic reticulum (ER) as a 57-kD proglutelin precursor, which is then processed into acidic and basic subunits in the protein storage vacuole. Three esp2 mutants, CM1787, EM44, and EM747, contain larger amounts of the 57-kD polypeptide and corresponding lower levels of acidic and basic glutelin subunits than normal. Electron microscopic observation revealed that esp2 contained normal-appearing glutelin-containing protein bodies (PB-II), but lacked the normal prolamin-containing PB (PB-I). Instead, numerous small ER-derived PBs of uniform size (0.5 microm in diameter) and low electron density were readily observed. Immunoblot analysis of purified subcellular fractions and immunocytochemistry at the electron microscopy level showed that these new PBs contained the 57-kD proglutelin precursor and prolamin polypeptides. The 57-kD proglutelin was extracted with 1% (v/v) lactic acid solution only after removal of cysteine-rich prolamin polypeptides, suggesting that these proteins form glutelin-prolamin aggregates via interchain disulfide bonds within the ER lumen. The endosperm of esp2 mutants contains the lumenal chaperones, binding protein and calnexin, but lacks protein disulfide isomerase (PDI) at the protein and RNA levels. The transcript of PDI was expressed in the seed only during the early stage of seed development in the wild type. These results suggest that PDI plays an essential role in the segregation of proglutelin and prolamin polypeptides within the ER lumen.

Deletion of the epidermal water/glycerol transporter aquaporin-3 (AQP3) in mice reduced superficial skin conductance by approximately 2-fold (Ma, T., Hara, M., Sougrat, R., Verbavatz, J. M., and Verkman, A. S. (2002) J. Biol. Chem. 277, 17147-17153), suggesting defective stratum corneum (SC) hydration. Here, we demonstrate significant impairment of skin hydration, elasticity, barrier recovery, and wound healing in AQP3 null mice in a hairless (SKH1) genetic background and investigate the cause of the functional defects by analysis of SC morphology and composition. Utilizing a novel (3)H(2)O distribution method, SC water content was reduced by approximately 50% in AQP3 null mice. Skin elasticity measured by cutometry was significantly reduced in AQP3 null mice with approximately 50% reductions in elasticity parameters Uf, Ue, and Ur. Although basal skin barrier function was not impaired, AQP3 deletion produced an approximately 2-fold delay in recovery of barrier function as measured by transepidermal water loss after tape stripping. Another biosynthetic skin function, wound healing, was also approximately 2-fold delayed by AQP3 deletion. By electron microscopy AQP3 deletion did not affect the structure of the unperturbed SC. The SC content of ions (Na(+), K(+), Ca(2+), Mg(2+)) and small solutes (urea, lactic acid, glucose) was not affected by AQP3 deletion nor was the absolute amount or profile of lipids and free amino acids. However, AQP3 deletion produced significant reductions in glycerol content in SC and epidermis (in nmol/microg protein: 5.5 +/- 0.4 versus 2.3 +/- 0.7 in SC; 0.037 +/- 0.007 versus 0.022 +/- 0.005 in epidermis) but not in dermis or blood. These results establish hydration, mechanical, and biosynthetic defects in skin of AQP3-deficient mice. The selective reduction in epidermal and SC glycerol content in AQP3 null mice may account for these defects, providing the first functional evidence for physiologically important glycerol transport by an aquaporin.

Glucansucrases are produced principally by Leuconostoc mesenteroides and oral Streptococcus species, but also by the lactic acid bacteria (Lactococci, Lactobacilli). They catalyse the synthesis of high molecular weight D-glucose polymers, named glucans, from sucrose. In the presence of efficient acceptors, they catalyse the synthesis of low molecular weight oligosaccharides. Glucosidic bond synthesis occurs without the mediation of nucleotide activated sugars and cofactors are not necessary. Glucansucrases have an industrial value because of the production of dextrans and oligosaccharides and a biological importance by their key role in the cariogenic process. They were identified more than 50 years ago. The first glucansucrase encoding gene was cloned more than 10 years ago. But the mechanism of their action remains incompletely understood. However, in order to synthesise oligosaccharides of biological interest or to develop vaccines against dental caries, elucidation of the factors determining the regiospecificity and the regioselectivity of glucansucrases is necessary. The cloning of glucansucrase encoding genes in addition to structure-function relationship studies have allowed the identification of important amino acid residues and have shown that glucansucrases are composed of two functional domains: a core region (ca. 1000 amino acids) involved in sucrose binding and splitting and a C-terminal domain (ca. 500 amino acids) composed of a series of tandem repeats involved in glucan binding. Enzymology studies have enabled different models for their action mechanism to be proposed. The use of secondary structure prediction has led to a clearer knowledge of structure-function relationships of glucansucrases. However, mainly due to the large size of these enzymes, data on the three-dimensional structure of glucansucrases (given by crystallography and modelling) remain necessary to clearly identify those features which determine function.

Lactic acid bacteria are among the most important probiotic microorganisms typically associated with the human gastrointestinal tract. Traditionally, lactic acid bacteria have been classified on the basis of phenotypic properties, eg, morphology, mode of glucose fermentation, growth at different temperatures, lactic acid configuration, and fermentation of various carbohydrates. Studies based on comparative 16S ribosomal RNA sequencing analysis, however, showed that some taxa generated on the basis of phenotypic features do not correspond with the suggested phylogenetic relations. Thus, some species are not readily distinguishable by phenotypic characteristics. This is especially true for the so-called Lactobacillus acidophilus group, the Lactobacillus casei and Lactobacillus paracasei group, and some bifidobacteria, strains of which have been introduced in many probiotic foods, eg, the novel yogurt-like commodities. Consequently, modern molecular techniques, including polymerase chain reaction-based and other genotyping methods, have become increasingly important for species identification or for the differentiation of probiotic strains. Probiotic strains are selected for potential application on the basis of particular physiologic and functional properties, some of which may be determined in vitro. The classification and identification of a probiotic strain may give a strong indication of its typical habitat and origin. The species, or even genus name, may also indicate the strain's safety and technical applicability for use in probiotic products. Molecular typing methods such as pulsed-field gel electrophoresis, repetitive polymerase chain reaction, and restriction fragment length polymorphism are extremely valuable for specific characterization and detection of such strains selected for application as probiotics.

Biodegradable polymers are designed to degrade upon disposal by the action of living organisms. Extraordinary progress has been made in the development of practical processes and products from polymers such as starch, cellulose, and lactic acid. The need to create alternative biodegradable water-soluble polymers for down-the-drain products such as detergents and cosmetics has taken on increasing importance. Consumers have, however, thus far attached little or no added value to the property of biodegradability, forcing industry to compete head-to-head on a cost-performance basis with existing familiar products. In addition, no suitable infrastructure for the disposal of biodegradable materials exists as yet.