Comparison of fumaric acid production by Rhizopus oryzae using different neutralizing agents

Fumaric acid fermentation in a 10-L bubble column fermenter using different neutralizing agents [CaCO(3), Ca(OH)(2), NaHCO(3)] by Rhizopus oryzae ATCC 20344 was examined. It was found that in the fermentation using CaCO(3 )as the neutralizing agent the highest fumaric acid weight yield and volumetric productivity were obtained, 53.4% and 1.03 g/L x h(-1) respectively. In the NaHCO(3) case, the fumaric acid weight yield and volumetric productivity were 33.7% and 0.69 g/L x h(-1), respectively, much lower than the CaCO(3) case. However, the NaHCO(3) alternative has advantages of cell reuse and simple downstream processing because of the high solubility of sodium fumarate. These advantages may offset the disadvantages of using NaHCO(3) as the neutralizing agent, and the overall fumaric acid weight yield and volumetric productivity will increase.     

Crystal structure of the cytoskeleton-associated protein glycine-rich (CAP-Gly) domain

Cytoskeleton-associated proteins (CAPs) are involved in the organization of microtubules and transportation of vesicles and organelles along the cytoskeletal network. A conserved motif, CAP-Gly, has been identified in a number of CAPs, including CLIP-170 and dynactins. The crystal structure of the CAP-Gly domain of Caenorhabditis elegans F53F4.3 protein, solved by single wavelength sulfur-anomalous phasing, revealed a novel protein fold containing three beta-sheets. The most conserved sequence, GKNDG, is located in two consecutive sharp turns on the surface, forming the entrance to a groove. Residues in the groove are highly conserved as measured from the information content of the aligned sequences. The C-terminal tail of another molecule in the crystal is bound in this groove.     

Biomass and aggregation analysis of human embryonic kidney 293 suspension cell cultures by particle size measurement

A method has been developed to monitor the cell growth of aggregated human embryonic kidney 293 (HEK293) suspension cultures by measuring cumulative particle volume and the particle size distribution. This method employs a particle size analyzer that determines the size of individual particles by detecting their light obscuration (blockage) or scattering. Cell counts derived from the cumulative volume of the cell particle correlate well with manual cell counts from a hemacytometer at different stages of growth. This correlation was further confirmed by quantifying total cellular protein of the samples. Simultaneously, the aggregation state of the samples can also be monitored and mathematically described. Results from this study demonstrate that this simple and reproducible method allows the direct measurement of cumulative cell volume and the degree of cell aggregation, as well as an indirect assessment of cell counts.     

Preservation of glucose metabolism in hypertrophic GLUT4-null hearts

GLUT4-null mice lacking the insulin-sensitive glucose transporter are not diabetic but do exhibit abnormalities in glucose and lipid metabolism. The most striking morphological consequence of ablating GLUT4 is cardiac hypertrophy. GLUT4-null hearts display characteristics of hypertrophy caused by hypertension. However, GLUT4-null mice have normal blood pressure and maintain a normal cardiac contractile protein profile. Unexpectedly, although they lack the predominant glucose transporter in the heart, GLUT4-null hearts transport glucose and synthesize glycogen at normal levels, but gene expression of rate-limiting enzymes involved in fatty acid oxidation is decreased. The GLUT4-null heart represents a unique model of hypertrophy that may be used to study the consequences of altered substrate utilization in normal and pathophysiological conditions.     

Protein coding gene trees in ciliates: comparison with rRNA-based phylogenies

We have reexamined the phylogeny of the ciliates using alpha-tubulin and phosphoglycerate kinase gene sequences. For alpha-tubulin, we have compared the amino acid and nucleotide sequences of 20 species representing seven of the nine classes of the phylum (Karyorelictea, Heterotrichea, Hypotrichea, Oligohymenophorea, Colpodea, Nassophorea, and Litostomatea). The phylogenetic tree resembles a bush from which three monophyletic lineages can be distinguished which correspond to the three classes Hypotrichea, Oligohymenophorea, and Litostomatea. For phosphoglycerate kinase, we have compared the amino acid sequences from 7 species representing three classes (Heterotrichea, Hypotrichea, and Oligohymenophorea). The branching pattern is resolved in three deeply separated branches with an early emergence of the heterotrich. Our comparative analysis shows that if alpha-tubulin phylogeny is not informative at the interclass level, the preliminary data from the phosphoglycerate kinase molecule appear more promising. Nevertheless, at low taxonomic level and at the class level, the resolved phylogenetic relationships inferred from both protein and rRNA sequence data are congruent.     

Furo[3′,2′:3,4]naphtho[1,2-d]imidazole derivatives as potential inhibitors of inflammatory factors in sepsis

Synthesis and anti-inflammatory effects of certain furo[3′,2′:3,4]naphtho[1,2-d]imidazole derivatives 12–18 were studied. These compounds were synthesized from naphtho[1,2-b]furan-4,5-dione (10) which in turn was prepared from the known 2-hydoxy-1,4-naphthoquinone (7) in a one pot reaction. Furo[3′,2′:3,4]naphtho[1,2-d]imidazole (12) was inactive (IC₅₀ value of >30 μM) while its 5-phenyl derivative 13, with an IC₅₀ value of 16.3 and 11.4 μM against lysozyme and β-glucuronidase release, respectively, was comparable to the positive trifluoperazine. The same potency was observed for 5-furan derivative 16 with an IC₅₀ value of 19.5 and 11.3 μM against lysozyme and β-glucuronidase release, respectively. An electron-withdrawing NO₂ substituted on 5-phenyl or 5-furanyl group led to the devoid of activity as in the cases of 14 and 17. Among them, compound 15 exhibited significant inhibitory effects, with an IC₅₀ value of 7.4 and 5.0 μM against lysozyme and β-glucuronidase release, respectively.For the LPS-induced NO production, the phenyl derivatives 12–15 were inactive while the nitrofuran counterparts 17 and 18 suppress LPS-induced NO production significantly, with an IC₅₀ value of 1.5 and 1.3 μM, respectively, which are more active than that of the positive 1400 W. Compounds 16–18 were capable of inhibiting LPS-induced iNOS protein expression at a dose-dependent manner in which compound 18, with an IC₅₀ of 0.52 μM in the inhibition of iNOS expression, is approximately fivefold more potent than that of the positive 1400 W. In the CLP rat animal model, compound 18 was found to be more active than the positive hydrocortisone in the inhibition of the iNOS mRNA expression in rat lung tissue. The sepsis-induced PGE2 production in rat serum decreased 150% by the pretreatment of 18 in a dose of 10 mg/kg.     

Rapamycin enriches for CD4(+) CD25(+) CD27(+) Foxp3(+) regulatory T cells in ex vivo-expanded CD25-enriched products from healthy donors and patients with multiple sclerosis

BACKGROUND: CD4(+) CD25(bright+) regulatory T cells (Treg) can be expanded to clinical doses using CD3/CD28 Ab-coated beads plus IL-2. However, this method requires high purity of the starting population to prevent overgrowth by non-regulatory T cells. Rapamycin, an agent that inhibits T-cell proliferation but selectively spares Treg, may be a means to expand Treg from less pure CD25-enriched cells. METHODS: CD25-enriched cells were prepared by a single-step immunomagnetic-selection using anti-CD25 microbeads. The cells were activated with a single addition of anti-CD3/CD28 beads and expanded in ex vivo 15-5% HS and autologous CD4(+) CD25(-) feeder cells,+/-rapamycin (0.01-20 ng/mL). IL-2 was added on day 3. Cells were rested for 2 days in ex vivo 15-5% HS and tested for phenotype, intracellular Foxp3 protein and suppressor activity. RESULTS: In the absence of rapamycin, CD25-enriched fractions expanded >17 000-fold by 21 days. Although suppressor activity was detected to day 14, it declined significantly by 21 days as non-regulatory cells expanded. The addition of rapamycin inhibited expansion of non-regulatory T cells at doses > or =1 ng/mL while increasing suppressor activity and the percentage of CD4(+) CD25(+) CD27(+) Foxp3(+) cells. Rapamycin did not enrich for Foxp3(+) cells in expanded cultures of CD4(+) CD25(-) cells. Treg were also readily expanded in cultures of CD25-enriched cells obtained from patients with multiple sclerosis in the presence of rapamycin. DISCUSSION: The addition of 1-20 ng/mL rapamycin to CD25-enriched cultures increased the purity of cells with the phenotype and function of Treg. This approach may alleviate the need for rigorous enrichment of Treg prior to activation and expansion for potential clinical use.     

Production of parvovirus B19 vaccine in insect cells co-infected with double baculoviruses

Recombinant human parvovirus B19 virus-like particles (VLPs), a candidate vaccine, were produced using the insect cell (Sf-9)-baculovirus (AcNPV) expression system. The synthesis and assembly of the particles in Sf-9 cells are directed by double infections with one recombinant virus (bacVP1) expressing the parvovirus minor viral protein VP1 and a second virus (bacVP2) expressing the major viral protein VP2. Previous animal studies demonstrated that the polypeptide composition of the VLPs strongly affects the elicitation of virus neutralizing antibodies. The key factor controlling the production of an immunologically potent product in bioreactors was identified to be the multiplicity of infection (MOI) of bacVP1 and bacVP2 used for infection. A probabilistic model, which correlates well with the experimental results, was employed to facilitate the selection of MOIs and to provide a better understanding of the baculovirus co-infection process. A novel production process based on secondary infections was developed to ensure product consistency and to simplify large-scale logistics. The effects of other critical process parameters, such as temperature, dissolved oxygen concentration, lactate concentration, cell concentration at infection, and harvest time, were also investigated. (c) 1996 John Wiley & Sons, Inc.