Microparticles prepared from biodegradable polyhydroxyalkanoates as matrix for encapsulation of cytostatic drug.

Microparticles made from degradable polyhydroxyalkanoates of different chemical compositions a homopolymer of 3-hydroxybutyric acid, copolymers of 3-hydroxybutyric and 4-hydroxybutyric acids (P3HB/4HB), 3-hydroxybutyric and 3-hydroxyvaleric acids (P3HB/3HV), 3-hydroxybutyric and 3-hydroxyhexanoic acids (P3HB/3HHx) were prepared using the solvent evaporation technique, from double emulsions. The study addresses the influence of the chemical compositions on the size and ξ-potential of microparticles. P3HB microparticles loaded with doxorubicin have been prepared and investigated. Their average diameter and ξ-potential have been found to be dependent upon the level of loading (1, 5, and 10 % of the polymer mass). Investigation of the in vitro drug release behavior showed that the total drug released from the microparticle into the medium increased with mass concentration of the drug. In this study mouse fibroblast NIH 3T3 cells were cultivated on PHA microparticles, and results of using fluorescent DAPI DNA stain, and MTT assay showed that microparticles prepared from PHAs of different chemical compositions did not exhibit cytotoxicity to cells cultured on them and proved to be highly biocompatible. Cell attachment and proliferation on PHA microparticles were similar to those on polystyrene. The cytostatic drug encapsulated in P3HB/3HV microparticles has been proven to be effective against HeLa tumor cells.

Biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) containing a predominant amount of 3-hydroxyvalerate by engineered Escherichia coli expressing propionate-CoA transferase.

AIMS: Of the biodegradable polyhydroxyalkanoates (PHAs), poly(hydroxybutyrate-co-hydroxyvalerate) (P(HB-co-HV)) is often considered for fabrication of biocompatible and absorbable medical devices and other applications. Depending on the application, however, specific mechanical or processing properties must be improved. To address these required properties, we sought to alter the monomer composition of the copolymer by a combination genetic engineering in an Escherichia coli host and carbon substrate feeding. METHODS AND RESULTS: We applied a new method of 3-hydroxyvalerate (3HV) monomer synthesis to produce a co-polymer by the introduction of a propionyl-CoA transferase gene (pct), along with PHA biosynthetic genes bktB, phaB and phaC from Ralstonia eutropha into engineered E. coli to produce P(HB-co-HV). The resulting strain successfully produced the copolymer containing an ultra-high 3HV monomer composition (over 80 wt%). CONCLUSIONS: To the best of our knowledge, the P(HB-co-HV) production strain constructed here synthesized polymer with the highest 3HV content of any engineered E. coli strain. This strain could also produce P(HB-co-HV) with the use of lower concentrations of propionate in the growth medium, compared to other reported strains, which could avoid the known growth inhibition from propionate in E. coli. SIGNIFICANCE AND IMPACT OF THE STUDY: Polyhydroxyalkanoates have been emphasized as a potential alternative for petroleum-based plastics by virtue of their physical properties and environmentally friendly characteristics. The copolymer produced in this work validates our genetic engineering approach and suggests that the Pct enzyme is a more efficient method for production of propionyl-CoA, the 3-hydroxyvaleryl-CoA precursor.

[Synthesis of 3-hydroxybutyrate-CO-4-hydroxybutyrate copolymers by hydrogen-oxidizing bacteria].

Synthesis of 3- and 4-hydroxybutyrate copolymer (3HB-CO-4HB), the most promising member of the biodegradable polyhydroxyalcanoate (PHA) family, has been studied. Cultivation conditions of naturally occurring strains of hydrogen-oxidizing bacteria Ralstonia eutropha B5786 and Cupriavidus eutrophus B10646 have been optimized to ensure efficient synthesis of the 3HB-CO-4HB copolymer. A set of highly pure samples of the 3HB-CO-4HB copolymer with 4HB content varying from 8.7 to 24.3 mol % has been obtained. Incorporation of 4-HB into the copolymer was shown to cause a more pronounced decrease in polymer crystallinity than the incorporation of 3-hydroxyvalerate or 3-hydroxyhexanoate; samples with a degree of crystallinity below 30% have been obtained. The weight average molecular mass of the 3HB-CO-4HB copolymers was shown to be independent on the monomer ratio and to vary broadly (from 540 to 1110 kDa).

The AP-1 site at -150 bp, but not the NF-kappa B site, is likely to represent the major target of protein kinase C in the interleukin 2 promoter.

Stimulation of T cells with antigen results in activation of several kinases, including protein kinase C (PKC), that may mediate the later induction of activation-related genes. We have examined the potential role of PKC in induction of the interleukin 2 (IL-2) gene in T cells stimulated through the T cell receptor/CD3 complex. We have previously shown that prolonged treatment of the untransformed T cell clone Ar-5 with phorbol esters results in downmodulation of the alpha and beta isozymes of PKC, and abrogates induction of IL-2 mRNA and protein. Here we show that phorbol ester treatment also abolishes induction of chloramphenicol acetyltransferase activity in Ar-5 cells transfected with a plasmid containing the IL-2 promoter linked to this reporter gene. The IL-2 promoter contains binding sites for nuclear factors including NFAT-1, Oct, NF-kappa B, and AP-1, which are all potentially sensitive to activation of PKC. We show that induction of a trimer of the NFAT and Oct sites is not sensitive to phorbol ester treatment, and that mutations in the NF-kappa B site have no effect on inducibility of the IL-2 promoter. In contrast, mutations in the AP-1 site located at -150 bp almost completely abrogate induction of the IL-2 promoter, and appearance of an inducible nuclear factor binding to this site is sensitive to PKC depletion. Moreover, cotransfections with c-fos and c-jun expression plasmids markedly enhance induction of the IL-2 promoter in minimally stimulated T cells. Our results indicate that the AP-1 site at -150 bp represents a major, if not the only, site of PKC responsiveness in the IL-2 promoter.

The Rhodococcus opacus PD630 heparin-binding hemagglutinin homolog TadA mediates lipid body formation.

Generally, prokaryotes store carbon as polyhydroxyalkanoate, starch, or glycogen. The Gram-positive actinomycete Rhodococcus opacus strain PD630 is noteworthy in that it stores carbon in the form of triacylglycerol (TAG). Several studies have demonstrated that R. opacus PD630 can accumulate up to 76% of its cell dry weight as TAG when grown under nitrogen-limiting conditions. While this process is well studied, the underlying molecular and biochemical mechanisms leading to TAG biosynthesis and subsequent storage are poorly understood. We designed a high-throughput genetic screening to identify genes and their products required for TAG biosynthesis and storage in R. opacus PD630. We identified a gene predicted to encode a putative heparin-binding hemagglutinin homolog, which we have termed tadA (triacylglycerol accumulation deficient), as being important for TAG accumulation. Kinetic studies of TAG accumulation in both the wild-type (WT) and mutant strains demonstrated that the tadA mutant accumulates 30 to 40% less TAG than the parental strain (WT). We observed that lipid bodies formed by the mutant strain were of a different size and shape than those of the WT. Characterization of TadA demonstrated that the protein is capable of binding heparin and of agglutinating purified lipid bodies. Finally, we observed that the TadA protein localizes to lipid bodies in R. opacus PD630 both in vivo and in vitro. Based on these data, we hypothesize that the TadA protein acts to aggregate small lipid bodies, found in cells during early stages of lipid storage, into larger lipid bodies and thus plays a key role in lipid body maturation in R. opacus PD630.

Biotechnology in food production and processing.

The food processing industry is the oldest and largest industry using biotechnological processes. Further development of food products and processes based on biotechnology depends upon the improvement of existing processes, such as fermentation, immobilized biocatalyst technology, and production of additives and processing aids, as well as the development of new opportunities for food biotechnology. Improvements are needed in the characterization, safety, and quality control of food materials, in processing methods, in waste conversion and utilization processes, and in currently used food microorganism and tissue culture systems. Also needed are fundamental studies of the structure-function relationship of food materials and of the cell physiology and biochemistry of raw materials.

Oxidized redox state of glutathione in the endoplasmic reticulum.

The redox state of the endoplasmic reticulum (ER) was measured with the peptide N-Acetyl-Asn-Tyr-Thr-Cys-NH2. The peptide diffused across cellular membranes; some became glycosylated and thus trapped within the secretory pathway, and its cysteine residue underwent reversible thiol-disulfide exchanges with the surrounding redox buffer. Glycosylated peptides from cells were disulfide-linked to glutathione, indicating that glutathione is the major redox buffer in the secretory pathway. The redox state of the secretory pathway was more oxidative than that of the cytosol; the ratio of reduced glutathione to the disulfide form (GSH/GSSG) within the secretory pathway ranged from 1:1 to 3:1, whereas the overall cellular GSH/GSSG ratio ranged from 30:1 to 100:1. Cytosolic glutathione was also transported into the lumen of microsomes in a cell-free system. Although how the ER maintains an oxidative environment is not known, these results suggest that the demonstrated preferential transport of GSSG compared to GSH into the ER lumen may contribute to this redox compartmentation.

PHA synthase activity controls the molecular weight and polydispersity of polyhydroxybutyrate in vivo.

A synthetic operon for polyhydroxyalkanoate (PHA) biosynthesis designed to yield high levels of PHA synthase activity in vivo was constructed by positioning a genetic fragment encoding beta-ketothiolase and acetoacetyl-CoA reductase behind a modified synthase gene containing an Escherichia coli promoter and ribosome binding site. Plasmids containing the synthetic operon and the native Alcaligenes eutrophus PHA operon were transformed into E. coli DH5 alpha and analyzed for polyhydroxybutyrate production. The molecular weight of polymer isolated from recombinant E. coli containing the modified synthase construct, determined by multiangle light scattering, was lower than that of the polymer from E. coli containing the native A. eutrophus operon. A further decrease in polyester molecular weight was observed with increased induction of the PHA biosynthetic genes in the synthetic operon. Comparison of the enzyme activity levels of PHA biosynthetic enzymes in a strain encoding the native operon with a strain possessing the synthetic operon indicates that the amount of polyhydroxyalkanoate synthase in a host organism plays a key role in controlling the molecular weight and the polydispersity of polymer.

Leveraging Industry-Academia Collaborations in Adaptive Biomedical Innovation.

Despite the rapid pace of biomedical innovation, research and development (R&D) productivity in the pharmaceutical industry has not improved broadly. Increasingly, firms need to leverage new approaches to product development and commercial execution, while maintaining adaptability to rapid changes in the marketplace and in biomedical science. Firms are also seeking ways to capture some of the talent, infrastructure, and innovation that depends on federal R&D investment. As a result, a major transition to external innovation is taking place across the industry. One example of these external innovation initiatives is the Sanofi-MIT Partnership, which provided seed funding to MIT investigators to develop novel solutions and approaches in areas of interest to Sanofi. These projects were highly collaborative, with information and materials flowing both ways. The relatively small amount of funding and short time frame of the awards built an adaptable and flexible process to advance translational science.

Oxidation of L-glucose by a Pseudomonad.

A new enzyme, D-threo-aldolse dehydrogenase (2S,3R-aldose dehydrogenase), found in Pseudomonas caryophylli, was capable of oxidizing L-glucose L-xylose, D-arabinose, and L-fucose in the presence of NAD+. The enzyme was synthesized constitutively and purified about 120-fold from D-glucose-grown cells. The Km values for L-glucose, L-xylose, D-arabinose, and L-fucose were 1.5 . 10(-2), 4.5 . 10(-3), 2.8 . 10(-3), and 2.1 . 10(-3), respectively. D-glucose and other aldoses inhibited the enzyme reaction; this inhibition was competitive with L-glucose as substrate and D-glucose as inhibitor. The optimum pH for the enzyme reaction was 10; the molecular weight of the enzyme was determined by gel filtration to be 7 . 10(4).

Puridication and properties of an intracellular ribonuclease from Candida lipolytica.

1. A ribonuclease (RNAase CL) (EC 3.1.4.23, ribonucleate 3'-oligonucleotide hydrolase) was extracted by EDTA/acetate buffer, pH 5.6 from acetonedried cells of Candida lipolytica and purified 1350-fold by acetone and (NH4)2SO4 fractionation, DEAE-cellulose and DEAE-Sephadex chromatography. 2. RNAase CL is an acidic protein having an isoelectric point of 4.2, and an approximate molecular weight of 32 000. 3. Optimal pH and temperature for the enzyme were 6.0 and 60 degrees C, respectively. It is stable at neutral pH up to 50 degrees C. At 64 degrees C for 30 min, 95, 49 and 64% inactivation of the enzyme occurred at pH values 4.2, 6.6 and 10.0, respectively. 4. RNAase CL inhibited by Zn2+ and Cu2+, sulfhydryl reactants and by high concentration of salts, but not by chelating agents. 5. RNAase CL degraded ribosomal RNA, transfer RNA, polyadenylic acid, polycytidylic acid and polyuridylic acid into acid-soluble nucleotides. Among the synthetic homopolymers, polycytidylic acid was most rapidly degraded. Polyguanylic acid and duplexes of synthetic homopolymers were less sensitive. DNA was not attacked. Specificity studies showed that RNAase CL preferentially cleaves pC-purine bonds. 6. Digestion of poly (C) by RNAase CL resulted in the liberation of cyclic 2',3'-CPM from the start of the reaction with no observable formation of intermediate oligonucleotides. This suggests that the enzyme depolymerizes by an exonucleolytic mechanism.

Metabolic engineering--methodologies and future prospects.

Attempts to improve the productivity of cellular systems or to increase metabolite yield often require radical alteration of the flux through primary metabolic pathways. However, achieving the desired result often proves difficult because the control architectures at key branch points have evolved to resist flux changes. Identification and characterization of these metabolic nodes is a prerequisite to rational metabolic engineering.

A C-terminal deletion in Corynebacterium glutamicum homoserine dehydrogenase abolishes allosteric inhibition by L-threonine.

In Escherichia coli, Bacillus subtilis and Corynebacterium glutamicum, homoserine dehydrogenase (HD), the enzyme after the branch point of the threonine/methionine and lysine biosynthetic pathways, is allosterically inhibited by L-threonine. To investigate the regulation of the C. glutamicum HD enzyme by L-threonine, the structural gene, hom, was mutated by UV irradiation of whole cells to obtain a deregulated allele, homdr. L-Threonine inhibits the wild-type (wt) enzyme with a Ki of 0.16 mM. The deregulated enzyme remains 80% active in the presence of 50 mM L-threonine. The homdr gene mutant was isolated and cloned in E. coli. In a C. glutamicum wt host background, but not in E. coli, the cloned homdr gene is genetically unstable. The cloned homdr gene is overexpressed tenfold in C. glutamicum and is active in the presence of over 60 mM L-threonine. Sequence analysis revealed that the homdr mutation is a single nucleotide (G1964) deletion in codon 429 within the hom reading frame. The resulting frame-shift mutation radically alters the structure of the C terminus, resulting in ten amino acid (aa) changes and a deletion of the last 7 aa relative to the wt protein. These observations suggest that the C terminus may be associated with the L-threonine allosteric response. The homdr mutation is unstable and probably deleterious to the cell. This may explain why only one mutation was obtained despite repeated mutagenesis.

Cloning and sequencing the Lactobacillus brevis gene encoding xylose isomerase.

The gene (xylA) coding for the Lactobacillus brevis xylose isomerase (Xi) has been isolated and its complete nucleotide sequence determined. L. brevis Xi was purified and the N-terminal sequence determined. All attempts to directly clone the intact xylA using a degenerative primer deduced from amino acids (aa) 10-14 were not successful. A fragment coding for the first 462 bp from the 5' end of xylA was isolated by PCR with two primers, one coding for aa M36 to W43 and the second coding for an aa sequence (WGGREG) conserved in a number of Xi's isolated from other bacteria. From the sequence of this fragment, two additional PCR primers were synthesized, which were used in an 'outward' reaction to clone a 546-bp fragment including a region upstream from the N terminus. Finally, the complete xylA gene was cloned in a 0.43-kb NlaIII-SalI fragment and a 1.9-kb SalI-EcoRI fragment. The 449-aa sequence for the L. brevis Xi shows homology with Xis isolated from other bacteria, especially within the primary catalytic domains of the enzyme.

Large-scale production of mammalian cells and their products: engineering principles and barriers to scale-up.

Mammalian cell products have great medical and clinical importance, but to date, production methods employed to manufacture these products on a large scale are not as cost efficient as they could be. The implementation of process control would greatly improve the productivity of these products. Recently developed methods to produce cells on a large scale, such as microcarriers, artificial capillaries, tubular spiral film, and microencapsulation must be optimized, and the problem of oxygen transfer limitation must be solved. The accumulation of potentially toxic waste products can inhibit growth and reduce productivity. This effect can be reduced by either adjusting the environmental parameters of a fed-batch culture, so that the cell's metabolism is shifted away from producing these compounds, or by continually perfusing medium through the culture. If these technical barriers can be overcome, the cost of producing products derived from mammalian cells can be greatly reduced.

Measurement and synthesis of insoluble and soluble dextran by Streptococcus mutans.

Total and insoluble dextransucrase activities of 10 strains of oral streptococci were measured by a modified filter disk assay. Strains that were nonadherent to hard surfaces had only low levels of insoluble dextransucrase activity. A physical rather than metabolic mechanism is suggested to explain the decreased insoluble and increased soluble activities observed when dextran T-10 is added to the media.

Continuous culture studies on the growth and physiology of Streptococcus mutans.

We examined the effects of nutritional limitations on the production of lactic acid by Streptococcus mutans grown at low growth rates in continuous culture. Lactic acid production was greater in nitrogen- and phosphate-limited continuous cultures than in glucose-limited conditions. These results are correlated with the release of calcium from enamel in cellfree broths from various fermentations.

The effect of different genetic properties of Salmonella typhimurium on the determination of stabilities and mutagenic concentrations of chemical carcinogens using the diffusion bioassay.

The mathematical model used to calculate half-life and mutagenic concentrations of chemical carcinogens from the diffusion bioassay does not include any terms related to the nature of the microorganism used in the assay (Awerbuch et al., 1979; Awerbuch and Sinskey, 1980). In this work we tested the model with different strains of Salmonella typhimurium. These strains are auxotrophs for histidine and are sensitive to base-pair substitution. The half-life (tau 1/2) of N-methylN'-nitro-N-nitrosoguanidine (NG) was calculated by the diffusion assay, using strains hisG46, TA1950, TA1535 and TA100 as the bacterial indicators. For all strains tau 1/2 equalled 2.2 h; strain sensitivity for detecting threshold mutagenic concentrations of NG was essentially the same, except that hisG46 was slightly more sensitive.

Quantitative determination of half-lifetimes and mutagenic concentrations of chemical carcinogens using a diffusion bioassay.

It is possible to deduce quantitative information about the mutagenic concentrations of direct carcinogens and procarcinogens from the well-known experiment in which a droplet of a chemical is put at the center of a petri dish containing a bacterial lawn in an agar gel. The model employed relates the mutagenic action to the space-time concentration profile of the diffusing substances. The range of mutagenic concentrations, in particular the lower limit Cmut, at which mutations occur, can be calculated from knowledge of the radii of the mutagenic zone. The only parameters necessary for the calculation of Cmut are the diffusion coefficient, D, which can be calculated, and the half-lifetime of the mutagenic substance, tau 1/2, which can be obtained from the diffusion experiments. The system was tested with N-methyl-N'-nitro-N-nitrosoguanidine (NG), N-methyl-N-nitrosourea (MNU), ethyl methanesulfonate (EMS), and acetoxydimethylnitrosoamine (AcDMN), which are direct carcinogens, and with nitrosomorpholine (NM) and nitrosopyrrolidine (NP), which are procarcinogens. As a consistency check, homogeneous experiments (dose-response curves) at low concentrations of the chemicals were also conducted. However, the statistical analysis of the results showed that the homogeneous assay is not adequate for determining threshold concentrations. Using the concept of the threshold to rank the tested chemicals according to mutagenic potency, the ranking is: NG greater than NP greater than MNU greater than AcDMN greater than EMS.

Redirection of carbon flux to lysine in a recombinant of Corynebacterium lactofermentum ATCC 21799 by limited supply of pantothenate.

To increase carbon flux to lysine, minimized production of amino acids that are biosynthetically related to lysine, for example, isoleucine and valine, is required. By limiting the supply of pantothenate, the precursor of coenzyme A, the carbon flux was redirected from isoleucine and valine to lysine in the recombinant of Corynebacterium lactofermentum ATCC 21799 containing the plasmid pGC77. The pGC77 contains hom(dr), thrB, and ilvA encoding feedback-deregulated homoserine dehydrogenase, homoserine kinase, and threonine dehydratase, respectively. At 250 microM of isopropyl-beta-d-thiogalactopyranoside, the recombinant (pGC77) produced lysine, valine, and isoleucine. Limiting the supply of pantothenate from 300 microg/l to 30 microg/l resulted in an increase in lysine (from 4.5 to 6.4 g/l) and decreases in valine (from 3.1 to 1.6 g/l) and isoleucine (from 0.9 to 0.3 g/l) production. The concentration of pyruvate was higher and that of acetate lower in the pantothenate-limited culture than in the control, suggesting that the limited supply of pantothenate delayed the conversion of pyruvate to acetyl-CoA. Increased availability of pyruvate by limiting the supply of pantothenate might favor the integration of pyruvate into the lysine branch. The results of this study are useful for the production of lysine with decreased concentrations of byproducts.